The levels of testosterone, dihydrotestosterone (DHT), 5.alpha.-androstane-3.alpha.-17.beta.-diol and androgen-binding protein (ABP) were measured in various segments of the epididymis from adult rats which had been unilaterally orchidectomized for 4 wk. On the intact side, ABP concentrations were highest in the caput region. The segmental distribution of DHT closely followed that of ABP with the highest concentration in the caput (40 ng/g tissue) and lowest in the cauda (10 ng/g tissue) epididymidis. There was a high degree of correlation (r = 0.98) between the concentration of DHT in the epididymis and ABP levels. Castration completely abolished the DHT gradient. The levels of testosterone and androstanediol were lower than those of DHT; most was present in the corpus epididymidis. The relative differences were reduced after castration. ABP in the rat epididymis is the primary factor for determining the concentration of DHT in the epididymal fluid.