1. A permeability barrier in or around the seminiferous tubules of rams has been demonstrated by studying the rate of passage of a variety of substances from blood plasma into fluid collected from the rete testis and into testicular lymph.2. All substances studied passed readily into testicular lymph.3. Tritiated water, urea, ethanol and bicarbonate in rete testis fluid equilibrated with blood plasma within 3 hr; Na(+), K(+), Rb(+), Cl(-), I(-), CNS(-), creatinine and galactose entered slowly and p-aminohippurate (PAH), glutamate, iodinated albumin, inulin and [(51)Cr]EDTA did not appear in rete testis fluid at all.4. Rubidium was excluded relative to iodoantipyrine from the testes of control and hypophysectomized rats and from rat testes heated to 37, 40, 43 and 45 degrees C; no such exclusion was seen in testes of rats which had been given cadmium chloride 5 months earlier so as to destroy the seminiferous tubules.5. It is suggested that this permeability barrier will regulate the access to the seminiferous epithelium of some constituents of blood plasma, isolate the germinal cells immunologically and help to maintain the concentration differences between rete testis fluid and lymph or blood plasma.