The specific binding of [125I]hCG to rat testicular membrane preparations was investigated as a function of membrane fluidity changed by lipids. Membrane fluidity was measured either by fluorescence depolarization of diphenylhexatriene or ESR spectra of I(1,14), I(12,3) and CAT 16 incorporated into the membrane. Incubation of membrane with cholesteryl-hemisuccinate increased both the rigidity of membrane lipids and the specific binding of [125I]hCG. A similar rigidifying action of saturated fatty acids was, however, not accompanied by increased accessibility of LH/hCG receptors. Treatment of testicular membranes with unsaturated fatty acids enhanced membrane fluidity but specific binding of the gonadotropin disappeared. In spite of the increase of LH/hCG receptors in cell membranes treated with cholesteryl-hemisuccinate, Leydig cells showed decreased sensitivity to cAMP response to LH stimulation. The results indicate that newly exposed LH/hCG receptors are not coupled with the adenylate cyclase system.