Outer dense fibres are located on the outside of the axoneme in the midpiece and principal piece of the mammalian sperm tail and may help to maintain the passive elastic structures and elastic recoil of the sperm tail. Here we describe the isolation and genomic organization of a rat gene encoding a cysteine-proline-rich outer dense fibre protein. The cDNA sequence of rts 5/1 and its expression pattern have already been published [Burfeind, P. & Hoyer-Fender, S. (1991) Dev. Biol. 148, 195-204]. There exist two different genes in the rat genome, rts 5/1 major and rts 5/1 minor. Rts 5/1 major consists of two exons interrupted by one intron of about 3.8 kb. Exon 1 and rts 5/1 minor contains a deletion of 120 bp, without destroying the open reading frame, which is flanked by short direct repeats, 15 bp in length. The first two nucleotides of the intronic sequence were identified as GA and, therefore, do not agree with the donor consensus sequence. From the analysis of mouse x rat cell hybrids, the rts 5/1 major gene has been assigned to chromosome 7. By immunoblotting and immunocytochemistry, it was demonstrated that the isolated gene encodes a major protein of rat sperm outer dense fibres.