Oligonucleotides offer the potential to inhibit the expression of specific genes within living cells by binding to the major groove of the DNA helix in a sequence-specific manner to form a DNA triple helix. The ultimate application of such oligonucleotides is to act as selective therapeutic tools to suppress transcription, inhibit replication and induce site specific mutations in mammalian genes. However, to date, most triplex experiments have utilised simple in vitro systems and it has proved rather more difficult to demonstrate selective actions in vivo. In this study, a series of psoralen-conjugated oligonucleotides (10-17 nt) were designed to target specific bovine repetitive sequences in the male Y chromosome. Repetitive Y chromosome sequences described by Reed et al., 1995 and Grobet et al., 1996 were investigated for efficient DNA triplex formation. The rationale in this approach was that the presence of several hundred copies rather than just one target per genome would greatly enhance the efficiency of DNA triplex formation in vivo. Psoralen-conjugated oligonucleotides designed to these sequences were screened by performing DNA triplex binding assays and analysing photoadduct formation by electrophoretic mobility shift assays (EMSA). Six psoralen-conjugated oligonucleotides were found to selectively and efficiently intercalate to their bovine dsDNA target sequences (38-49 bp) in vitro after irradiation with UV light (360 nm), forming covalent DNA photoadducts. Stable photoadducts were also detected when these oligonucleotides were targeted to larger dsDNA targets, ranging from 0.1 to 3 Kb in size. In general, a small UV dose (0.03 J/cm²) was sufficient to form monoadducts, with crosslinks forming after fifteen minutes of UV irradiation (1 J/cm²). The efficiency of bovine monoadduct formation was similar to that in the well-publicised supF DNA triplex system. PCR and Southern blot analyses also showed that the repetitive Y-chromosomal DNA region (100-400 bp) was indeed male specific and present in multiple copies. However, when Southern blot analyses were carried out using oligonucleotide probes (16-49 nt) which corresponded to the triplex target sequence within this region, relatively weak hybridisation was observed, suggesting that these specific sequences were not present in such high copies. Although this data indicates that the male specific repeat regions must contain segments of consensual rather than exact sequences, high quality non-degenerate DNA sequencing data in the triplex targeting region was obtained from the male specific PCR products. Clearly, the exact nature of these male specific repeats requires further investigation to establish the effects of targeting psoralen-conjugated oligonucleotides to male genomic DNA as opposed to female genomic DNA.