IN mammals spermatozoa mature while passing through the epididymis. In the rabbit there are virtually no fertile spermatozoa in the caput epididymidis and fertilizing ability is acquired when, the spermatozoa pass through the corpus epididymidis1,2. Spermatozoa retained by ligatures, in the proximal corpus develop a normal fertilizing ability in 24 h, but never become fertile when retained for 1 to 12 days in the proximal caput epididymidis3,4. These results strongly suggest that the factors governing the maturation process are not intrinsic to the spermatozoa. To determine whether the development of the fertilizing ability can be achieved similarly when the proximal corpus is maintained 24 h in vitro in organ culture, we used the technique described below. Testes were reached aseptically by means of an abdominal incision, and connective tissue and fat were gently peeled from the epididymis of the anaesthetized animal. The tissue was then removed; the proximal corpus and distal corpus were separated and each placed in 1 ml. of a solution of 0.05% pronase or of 0.5% clostridium peptidase for 30 to 60 min at 31° C. The epididymal tubule was gently unconvoluted and placed unbroken on an agar strip. Trowell's5 organ culture technique was used with Steinberger's6 modification. The medium was Eagle's7 minimal medium with 15% foetal calf serum and 100 I.U. penicillin-streptomycin mixture. The gas phase was air containing 5% CO2, and the cultures were incubated at 31° C, pH 7.0±0.2. A 2 mm segment from the epididymis just distal to the distal corpus was cut up in 0.5 ml. of the same tissue culture medium. The sperm suspension was incubated in a test-tube for 24 h under the same conditions. After 24 h the epididymal tubule placed in organ culture was removed, cut up in 1 ml. Hank's solution and the debris removed. The two sperm suspensions were injected through both uterine walls of one or two does, which were injected with an ovulatory dose of 50 I.U. of human chorionic gonadotrophin (HCG) at the time of insemination. Females were killed 26 h after injection of HCG. The oviducts were flushed and the eggs recovered were examined for the presence of pronuclei or of cleavage, taken as evidence of fertilization. Nuclear details were clarified with acetocarmine staining after fixation in 1:3 acetic alcohol8. The male was then killed, the contralateral testis and epididymis removed and the proximal corpus and distal corpus were separated, cut up in 0.5 ml Hank's solution and the debris removed. Sperm suspensions were inseminated in 1 or 2 does as described above. A total of 13 males and 58 females were used.