The mechanisms involved in the maintenance of the endocrinological microenvironment of the seminiferous and epididymal tubules were examined in a series of experiments utilizing in vivo microperifusion, microperfusion, and micropuncture technique. The intraluminal 3H-androgen concentration in the seminiferous tubules increased linearly as the interstitial 3H-androgen concentrations increased from 10 nM to 2,000 nM, but in the caput epididymidal tubules, the intraluminal 3H-androgen concentration increased hyperbolically across the same range of peritubular 3H-androgen concentration. Intraluminal 3H-androgen concentrations in the caput epididymidis did not rise above approximately 340 nM even when the peritubular 3H-androgen concentration exceeded 2,000 nM. Perifusion of caput tubules with 0.1 mM dinitrophenol or potassium cyanide or 100 micrograms/ml cyclohexamide significantly reduced the proluminal 3H-androgen movement, but tubules perifused with control medium did not support antigrade 3H-androgen movement in the absence of native lumen fluids which contain androgen-binding protein. Energy-requiring protein synthesis is necessary for antigrade 3H-androgen movement in the caput epididymidis, but the mechanism for the interaction of intracellular protein(s) and 3H-androgen movement remains undetermined.