Selenium is necessary for normal sperm tail development in the rat. The biochemical locus of this requirement was investigated by intratesticular administration of (75Se)SeO3-2. Sperm were labeled primarily in tail keratin, a disulfide-stabilized fraction obtained by extracting isolated tails with sodium dodecyl sulfate (SDS). By incubation in 1% SDS-0.2 mM dithiothreitol, followed by sonication and centrifugation, the 75Se was separated from a residue rich in S and Zn, composed of dense fibers and connecting pieces. The extracted 75Se-labeled material was non-dialyzable, sensitive to pronase, retained most of its bound isotope following carboxymethylation, and displayed only one radioactive component, of 17,000 daltons, during SDS-polyacrylamide gel electrophoresis. It is suggested that the labeled moiety is a selenopolypeptide whose function is critical for normal assembly of the sperm tail.