A cytoplasmic receptor for testosterone (T) was characterized in Sertoli cell-enriched (SCE) testes from hypophysectomized (hypex) rats. Receptor binding was also measured in tubules isolated from SCE testes and primary cultures of Sertoli cells (33 fmol/106 cells). Receptors labeled at 0.degree. C with [3H]T .+-. excess unlabeled T was precipitated with ammonium sulfate and specific binding was measured by gel filtration. Specific binding reached equilibrium within 2 h and maximum binding was achieved at 3-6 nM [3H]T. Scatchard analysis revealed a Kd of 7.6 .times. 10-10 M which was similar to receptor preparations from non-irradiated hypex rats (6.8 .times. 10-10 M). The specific activity of receptors in non-irradiated hypex testis (12.7 fmol/mg prot) was greater than that in SCE hypex testis (9.6 fmol/mg prot [protein]). The receptor could be precipitated quantitatively at 40% ammonium sulfate, thereby separating ABP which precipitated at 50% and albumin which precipitated between 60 and 80%. The receptor was labile to heat, charcoal and sulfhydryl reagents. It had a t1/2 of several days, was excluded by Sephadex G-200, and migrated slower than ABP during polyacrylamide gel electrophoresis. Relative affinities for steriods were T > DHT [dihhydrotestosterone], 5.alpha.-androstane-3.alpha., 17.beta.-diol > cyproterone acetate, 5.alpha.-androstane-3.beta., 17.beta.-diol > androstenedione, dehydroepiandrosterone, cortisol.