The present study was undertaken to determine whether or not antigrade, proluminal 3H-androgen movement in the caput epididymis occurs in the absence of native lumen content. A single tubule was perfused with artificial caput fluid containing no androgen-binding protein for 30 min and subsequently tubules were perifused with Minimum Essential Medium perifusion fluid containing 26.7 microCi/ml 3H-testosterone and 1.3 microCi/ml 14C-polyethyleneglycol for 1 h. Radioactivity of isotopes in perifusion and intraluminal fluids was determined at 1 h after sustaining perifusion, and the percentage of peritubular isotopes appearing in the intraluminal fluid was determined. Net entry of 3H-androgen into the epididymal tubules in the presence of native intraluminal content was approximately 323%. In contrast, intraluminal 3H-androgen concentrations in the epididymal fluid in the absence of native lumen content were significantly reduced, to 100% of those in the peritubular fluid. Antigrade, proluminal movement of 3H-androgen in the caput epididymis does not occur in the absence of native lumen content. Androgen-binding protein in the epididymal lumen may be required to maintain uphill proluminal movement of 3H-androgen into the tubules.