The distribution of [3H] carnitine in the epididymis has been studied by autoradiography 93 hours after injection of [3H]deoxycarnitine into intact, hemicastrated or hypophysectomized rats. In the intact animals, radioactivity is concentrated in the lumen of the caput and corpus with only a small amount observed in the caudal portion of the epididymis. In the hemicastrated rat, devoid of spermatozoa, the amount of radioactivity in the lumen of the caput and the cauda portions are lower than in the intact rats but still greater compared to the epididymal cells. In hypophysectomized rats the luminal labelling is much reduced. In contrast to the epididymis, the amount of radioactivity is low and evenly distributed in the heart from intact, hemicastrated and hypophysectomized rats and in the pituitary from intact and hemicastrated animals. The great reduction in luminal labelling after hypophysectomy emphasizes the hormonal dependence of the carnitine concentrating mechanism. These findings indicate that carnitine initially is concentrated in the caput and corpus portion of the epididymis. This intraluminal carnitine is subsequently transported into the caudal segment.The evidence strongly supports the existence of a hormonal dependent carnitine concentrating mechanism localized on the luminal side of the epithelium in epididymis.