Testis proteins of Drosophila melanogaster deficient for six different Y-chromosome regions were fractionated by means of a sodium dodecyl sulfate/polyacrylamide gel system designed to separate high molecular weight polypeptides (Mr, greater than 200,000). Analysis of the banding patterns indicates that the three regions containing fertility genes kl-2, kl-3, and kl-5 are responsible for three different high molecular weight polypeptides. Several observations indicate that these polypeptides are structural components of the sperm axoneme. They are present in seminal vesicles, which are highly enriched for mature sperm. They are first detected during development at a time when the first spermatids are elongating. Finally, deletion of either kl-5 or kl-3 leads to the absence of the outer dynein arm of the peripheral doublets of the axoneme. Although absence of the kl-2 region eliminates the third polypeptide, an associated structural defect in the axoneme has yet to be identified. The three polypeptides are in the Mr 300,000-350,000 range, and their mobilities are similar to those of dynein polypeptides from Chlamydomonas axonemes. Experiments using dosage variation and a temperature-sensitive sterile mutation in kl-5 suggest that the Y-chromosome regions contain the coding sequences for the polypeptides.