The presence of prion protein in sperm and fluids collected from different parts of the ram genital tract was investigated by immunoblotting with monoclonal antibodies. A slightly immunoreactive 25- to 30-kDa protein was recognized on Western blots of testicular and epididymal sperm extracts. Immunoreactivity increased on ejaculated sperm extracts and 2 other bands at 35 and 43 kDa also reacted. Seminal plasma showed several immunoreactive bands, the main bands being detected at 43 and 35 kDa, whereas less reactive bands were observed at 30, 25, 20, and <14 kDa. All these bands strongly decreased in the seminal plasma after vasectomy, indicating a testicular or an epididymal origin. Testicular fluid showed almost no reactivity, whereas caudal epididymal fluid contained the 2 strong immunoreactive bands at 43 and 35 kDa and in some cases a faint 30-kDa band. The 43-kDa band was also found in the fluid from the proximal caput, whereas the 35-kDa band appeared in the distal caput. Immunoprecipitation of S-35-labeled proteins secreted in the epididymal fluid indicated that the 43-kDa form was synthesized in caput and caudal regions and the 35-kDa form in the distal caput to the distal corpus. Treatment of caudal fluid and seminal plasma by N-glycosidase resulted in the formation of 3 bands: 1 highly reactive at about 25 kDa, a second less reactive at about 28 kDa, and a third at approximately 20 kDa. The pattern of prion protein distribution in epididymal fluids was found to be similar in scrapie-infected rams to that of healthy rams. Cauda epididymal fluid and seminal plasma from infected animals could not be treated directly with proteinase K. because of the presence of protease inhibitors. However, the prion protein immunoprecipitated from these fluids was completely cleaved by proteinase K, whereas in the same conditions this from an infected sheep brain gave the usual resistant band pattern.