The mechanism whereby glycerol exerts its antispermatogenic action is not known. The objective of this study was to determine, with the use of [3H]inulin and [125I]albumin by in vivo and in vitro methods, whether glycerol exerts its effect by altering the permeability of the blood-testis barrier (BTB). Adult male rats received a single intratesticular injection of either glycerol (10% or 20%; treated) or saline (control), and 2, 4, 8, 26, and 56 weeks after treatment, either [3H]inulin or [125I]albumin was administered either by infusion or directly to the testicular tissues. Radioactivity was measured in testicular tissue, rete testis fluid, and seminiferous tubular fluid. Following in vivo administration, the uptake of [3H]inulin by seminiferous tubules, rete testis fluid, and seminiferous tubule fluid was significantly greater in the treated than in the control testes at all times after treatment. Radiolabeled inulin, injected into isolated testes or added to medium in which isolated tubules were incubated, accumulated at significantly higher levels in the seminiferous tubule compartment of treated than control tissues. Rete testis fluid from treated testes, collected by micropuncture following efferent duct ligation, contained about 5- to 13-fold more radioactivity than fluid from controls. Following infusion of 50 microCi of [125I]albumin into the jugular vein, the accumulated radioactivity was significantly greater in testicular and epididymal tissues and in the seminiferous tubule fluid from treated than from control animals. In all experiments the significant differences between treated and control were maintained during the period of 2-56 weeks following glycerol treatment.(ABSTRACT TRUNCATED AT 250 WORDS)