In order to facilitate the biochemical analysis of spermatogenesis in the nematode Caenorhabditis elegans methods have been developed for obtaining large quantities of males and for the isolation of sperm. Males are isolated by a passive filtration method from strains producing high proportions of males and sperm are isolated by physical pressure followed by filtration and differential centrifugation. Biochemical analyses show that sperm contain a major protein component that represents 17% of the total sperm protein. This protein has a molecular weight of 15,600, an isoelectric pH of 8.6, and exists as a dimer. It is shown by immunocytochemical techniques to be a specific product of spermatogenesis. It is localized in the proximal arm of the male gonad and in the sperm of both the male and hermaphrodite but it is not detected in other tissues of the nematode. It is not a nuclear binding protein. Pulse-labeling studies show that this major sperm protein is first synthesized in the proximal arm of the male gonad beginning at 39-42 hr after hatching at 20 degrees C. Poly(A) mRNA coding for this protein is first detected in a translatable form just before synthesis of this sperm protein suggesting transcriptional control.