The incorporation of radioactive mannose and fucose into secretory glycoproteins by rat epididymal tissue was studied using tissue pieces in vitro. The appearance of radioactive macromolecular products in the medium occurred after a lag phase of 2 h with radioactive mannose, but with radioactive fucose the lag phase was only 15 min. Preincubation of tissue for 2 h before the addition of radioactive mannose increased the subsequent rate of incorporation by reducing the lag phase from 2 to 1 h. Tunicamycin reduced the incorporation of radioactive mannose and fucose into macromolecular products to approximately 15 and 50% of normal in the caput and cauda respectively; maximum inhibition required 10 micrograms tunicamycin/ml in the caput and 2 micrograms/ml in the cauda. Reduction of radioactive sugar incorporation by tunicamycin did not result in qualitative changes in the profile of the radioactive glycoproteins that were secreted. However, immunoprecipitation of proteins D and E from incubations with radioactive methionine or mannose revealed that tunicamycin caused these proteins to be synthesized and secreted in a non-glycosylated form. Prior castration of animals reduced the incorporation of radioactive mannose and fucose, and qualitative changes in the profiles of secreted radioactive glycoproteins were apparent.