Antibodies against ubiquitin, a universal proteolytic marker, show increased cross-reactivity with defective spermatozoa in men and bulls. We investigated sperm ubiquitination in the stallion, a seasonally polyestrous mammal. Immunofluorescence and immunoelectron microscopy demonstrated that anti-ubiquitin antibodies bind to the surface of both membrane-intact and aldehyde-fixed spermatozoa. Cross-reactivity to the ubiquitin-conjugating enzyme E2 was also detected in sperm. Immunohistochemistry showed that ubiquitinated spermatozoa were first detected in the caput epididymis, coincident with a strong accumulation of ubiquitin and ubiquitin C-terminal hydrolase, protein gene product 9.5, in the apical stereocilia of the epididymal epithelium. Testicular spermatozoa did not display significant ubiquitin cross-reactivity. Similarly, lesser accumulation of ubiquitin cross-reactive substrates was identified in the accessory sex glands. Semen samples were collected from three fertile stallions and one subfertile stallion between December and February and probed for ubiquitin by flow cytometry and immunoblotting. Flow cytometric analysis showed that sperm from the subfertile stallion had higher ubiquitin levels than sperm from the other three stallions. In addition, immunoblot analysis of sperm proteins from the subfertile stallion showed two unique ubiquitin cross-reactive bands that were not present in sperm extracts from the three fertile stallions. To screen for a possible role for ubiquitin in seasonal changes in sperm production, semen samples from two fertile stallions were collected in March, June, September, and December and subjected to a flow cytometric ubiquitin assay. The lowest levels of ubiquitin-labeled sperm were found in March, approximately coincident with the onset of the natural horse breeding season. A progressive increase in sperm ubiquitin levels was found during summer and fall, with a peak in December. These data suggest that stallion sperm are differentially ubiquitinated during epididymal maturation and that this ubiquitination may reflect changes in sperm numbers and semen quality. The association between changes in sperm ubiquitination and seasonal changes in sperm production will be subjected to further studies in a larger cohort of animals.