The binding of human sex hormone-binding globulin (hSHBG) to plasma membranes prepared from the adult rat epididymis and other potential target and non-target tissues was examined. Specific binding sites were detected in the epididymis, testis, prostate, skeletal muscle and liver. The first three organs exhibited a higher (KD approx. 0.1 nM; Bmax approx. 0.05-0.10 pmol/mg membrane protein, Site I) and a lower (KD approx. 5 nM; Bmax approx. 1.0-2.5 pmol/mg membrane protein, Site II) affinity binding site. Only Site I was detected in muscle membranes and only Site II was detected in membranes isolated from liver. Specific binding was not detectable in either spleen or brain. Regional distribution of hSHBG binding sites occurred in the epididymis. Both Site I and Site II were present in the proximal caput and distal cauda. The distal caput and proximal cauda contained only Site II; no specific binding was detected in the corpus. Binding of hSHBG to epididymal membranes was time- and temperature-dependent. The presence of Ca2+ did not affect binding. Non-liganded [125I]-labeled hSHBG can bind to both sites in epididymal membranes. The affinity of hSHBG for Site I increased 2-fold when it was complexed with 5 alpha-dihydrotestosterone, testosterone or estradiol. The hSHBG-androgen complex had little effect on Site II versus steroid-free SHBG. However, the affinity of the hSHBG-estradiol complex for these sites was increased 10-fold. Cortisol, which has a low affinity for hSHBG, did not influence its binding to either the higher or lower affinity membrane sites.