The fertilizing ability of spermatozoa from epididymal tubules maintained in organ cultures from 1 to 7 days was assessed after artificial insemination into receptive does. It was found that spermatozoa from the distal corpus which were already capable of fertilizing eggs prior to the cultures retain this ability for 1 day without addition of hormone and for 3-4 days when testosterone (0.5 mug/ml) or 5alpha-dihydrotestosterone (0.5 mug/ml) is added to the culture medium. Spermatozoa from the proximal corpus which were not capable of fertilizing eggs prior to the cultures remain so after 1 day in cultures without addition of hormone. Testosterone, 5alpha-dihydrotesterone, 3alpha-androstanediol, or 3beta-androstanediol was added to cultures of proximal corpus at a concentration of 0.5 mug/ml. Only with 5alpha-DHT is the mean percentage of fertilization significantly higher than the percentage obtained without addition of hormone. Insulin does not potentiate the effect of 5alpha-DHT on sperm fertilizing ability. Epithelial growth factor is ineffective. Spermatozoa from the caput epididymidis kept in cultures for 1 to 4 days remain infertile. The results are discussed in light of the morphological findings presented in the preceding communication and in relation to the physiological requirement for sperm maturation in the epididymis.