The outer dense fibres (ODF) are a main cytoskeletal structure of the sperm tail. Despite their importance in the morphology and function of the sperm tail, their constituents are poorly described. Here we investigate the protein composition of human outer dense fibres. Our results suggest that human ODF consist of about 10 major and of at least 15 minor proteins, where all major proteins are ODF1, ODF2 or ODF2-related proteins. From a human testis cDNA library, we isolated two slightly different cDNAs encoding ODF2 proteins of approximately 70 kDa. Human ODF2 cDNAs and their encoded proteins are very similar to those isolated from rat and mouse pointing to a high evolutionary pressure residing on these proteins. Transcription of ODF2 is restricted to testis tissue and more specifically to round spermatids as was demonstrated by a non-radioactive in-situ hybridization. ODF2 proteins were detected in the sperm tail. Their distribution along the length of the sperm tail shows that the ODF normally extend to about half the principal piece of the sperm tail. The former result opens the possibility for a screening regarding the distribution of sperm tail proteins related to motility disorders.