We have been able to culture caput epididymal tubules from rats by modifying an organ culture system (Orgebin-Crist et al, 1980) which was used to culture rabbit corpus epididymal tubule segments. The morphology of the epithelium was consistently good throughout seven days in culture, although sloughed epithelium was commonly seen during the first 24 hours. Evidence of this sloughing was much less frequent thereafter. Throughout seven days of culture, autoradiography of SDS-PAGE of luminal fluid obtained from tubules cultured in medium containing 14C-L-leucine showed incorporation into bands identical to those stained by Coomassie Blue. Rat epididymal alpha-lactalbumin was consistently localized on the luminal surface of the epithelium and the middle piece of the spermatozoa. Spermatozoa appeared to have normal morphology throughout the first three days in culture; thereafter, decapitated spermatozoa were frequently seen. Caput spermatozoa only quiver in place prior to culture, but after three days in culture, 53% of the spermatozoa from distal caput tubules are progressively motile upon dilution in a balanced salt solution. Since the transit time for spermatozoa in the caput epididymidis of the rat is approximately three days, it should be possible with this culture system to study maturational events involving interactions between spermatozoa and the epididymal epithelium.