Methanotrophic populations have been studied in Newport Bay estuary, Southern California. Environmental clone banks were generated for 16S rRNA genes specific to methanotrophs and for a diagnostic functional gene, pmoA, encoding a conserved subunit of the particulate methane monooxygenase. These clone banks contained sequences specific to types I and II methanotrophs typically found in aquatic environments including freshwater lake and soda lake sediments, aquifers and rice paddies. However, a group of clones that were divergent (93% identity) from known methanotrophic 16S rRNA genes but represented in 16S rRNA gene libraries from other aquatic environments were detected. A group of pmoA sequences divergent (83% identity) from extant methanotrophs and not previously represented in any environmental clone libraries, were also detected. It is concluded that this environment contains significant methanotroph diversity and that some of these may represent novel groups of methanotrophic bacteria.