Traditionally, culture-based approaches have been used to investigate the microbial ecology of thermal habitats. The emergence of alternate molecular approaches based on DNA sequences has shown that terrestrial thermal environments have a rich diversity of uncultured prokaryotes. To the best of our knowledge these latter approaches have not previously been applied to New Zealand thermal environments and this became the central aim of this study. The major objective of the study was to target the metabolically active members of the hot pool community by applying a suite of molecular and microscopical techniques to those species colonising surfaces incubated in situ in the hot pools. Two neutral pH pools were investigated in some detail; pool KP1 at 75°C and pool AQ1 at 95°C. Surfaces in pool KP1 were colonised by a high diversity of bacteria (archaea were not detected), as evidenced by complex denaturing gradient gel electrophoresis (DGGE) profiles, and many different species were obtained in enrichment culture. Although colonisation rates were greater in pool AQ1, this was entirely due to only two observed morphotypes and was reflected in DGGE profiles that reflected only two dominant archaeal species. A 16S rDNA clone library of the AQ1 pool water and DGGE profiles of both pool water and colonised slides were dominated by rods of the species Pyrobaculum. Slides incubated deep within AQ1 were also colonised by cocci, the closest cultured relative being Aeropyrum pernix, a marine hyperthermophile. Two pure cultures were obtained from pool AQ1, Pyrobaculum sp. (AQ1.S2) and a novel crenarchaeaotal coccus, (AQ1.S1T). Isolations of the latter organism were also obtained from two other pools in New Zealand. These were obtained as obligate co-cultures with Pyrobaculum rods and could not be isolated into pure culture. A full description of this novel genus of organisms has been accepted for publication in the International Journal of Systematic and Evolutionary Microbiology, with the isolate from pool AQ1 named as the type strain lgnisphaera aggregans AQl .S1T. The techniques used to follow colonisation were then applied to a survey of high-temperature pools of New Zealand (>80°C, pH 5.5-9.7), White Island (72-94°C, pH 1.5-3.8) and Yellowstone National Park (75-92.5°C, pH 5.0-8.0). For New Zealand, bacteria were detected by DGGE on colonised slides in pools below 88°C, and archaea, only in pools above 83°C. Bacterial colonisers with dominant signatures included those of Thermodesulfobacteria and Aquificales species, though interestingly, isolations of these organisms were not obtained. The archaeal colonisers were pH dependent: pools with a pH of 6.0-7.0 had a dominant Thermofilum signature; pools above pH 7.0 had both a Pyrobaculum sp. and a relative of the Desulfurococcales; and the acidic pools (including White Island) were dominated by Sulfolobus sp. Pool water DGGE profiles of the New Zealand pools differed from slide colonisation profiles in some respects, e.g. Thermococcus sp. were only detected in pool water. In contrast to the New Zealand results, the Yellowstone National Park slides were dominated by bacteria, with bacterial signatures detected in high-temperature pools (87-92.5°C); representative DGGE bands were sequenced and found to be related to the Aquificales. Nanoarchaeal l 6S rDNA PCR amplicons were also obtained from pool water for most of the New Zealand pools (closest relative of AQ1 amplicon being a nanoarchaeal clone from the Uzon Caldera), including the Pyrobaculum sp. AQl .S2 culture. Attempts were also made to isolate marine nanoarchaea from nascent hydrothermal vents situated on the East Pacific Rise; however, these were unsuccessful.