The effect of metabolic inhibitors on transepithelial movement of 3H-androgens was investigated by in vivo perifusion and subsequent micropuncture of caput and cauda epididymal tubules. Epididymal tissue (adenosine triphosphate (ATP) concentrations were determined at 1 h after exposure of perifusion fluid with metabolic inhibitor. To determine whether or not metabolic inhibitor alters intraluminal androgen-binding protein concentration or androgen binding to interstitial proteins in the caput epididymis, 3H-dihydrotestosterone (DHT) binding to interaluminal androgen-binding protein and bound vs free androgen ratio in the interstitial fluid after 1 h perifusion with fluid containing metabolic inhibitor around caput epididymal tubules were examined. Proluminal movement of 3H-androgens and tissue ATP concentrations in the caput and cauda epididymis were significantly decreased by addition of dinitrophenol (DNP) or potassium cyanide to the perifusion fluid. Relative intraluminal androgen-binding protein protein concentration and bound vs free 3H-androgen ratio in the interstitial fluid were not altered when DNP or potassium cyanide was added to the perifusion fluid. These results demonstrate for the first time that an energy-dependent mechanism may be involved in the epididymal androgen uptake.