The binding of ring B unsaturated equine estrogens, equilin sulfate (EqS), equilin (Eq), and 17 beta-dihydroequilin (17 beta-Eq) with human serum proteins was determined and compared with the binding of estrone sulfate (E1S), estrone (E1), estradiol (E2), testosterone (T), and 5 alpha-dihydrotestosterone (5 alpha-DHT). Undiluted serum or 5% human serum albumin (HSA) was incubated with 3H-labeled steroids at 37 C, then subjected to gel filtration at 4 C. Gel filtration of serum from Premarin-treated postmenopausal women or normal women incubated with Eq, E1, E2, or 5 alpha-DHT showed two peaks of radioactivity associated with proteins with average apparent mol wt of 128,000 and 68,000 and average Stokes radii of 48.6 and 34.9 A. These values correspond to those reported for sex hormone-binding globulin (SHBG) and albumin, respectively. Binding to SHBG and albumin was confirmed by removing SHBG or albumin from the serum with Concanavalin-A Sepharose 4B gel or CM-Affi Gel Blue, respectively. In the case of [3H]EqS and [3H]E1S, binding to SHBG was not detectable, and only a peak of radioactivity associated with albumin was found. However, under these conditions, the binding of estrogens to SHBG in serum from normal men was not detectable. Incubation of the above steroids with 5% HSA followed by gel filtration resulted in a single peak of radioactivity associated with the protein peak. Using ultrafiltration dialysis followed by Scatchard analysis, at least two sets of binding sites were found for the interaction of HSA with EqS or E1S. The high and low affinity binding sites had association constants k1 and k2 of approximately 0.9-1.1 (X 10(5) M-1) and 0.5-0.8 (X 10(4) M-1). In contrast with Eq and E1, only the low affinity binding sites were found (apparent Ka congruent to 1 X 10(4) M-1). The binding constants of some estrogens and androgens to SHBG at 37 C determined by competitive Scatchard analysis using DEAE filter assay and [3H]5 alpha-DHT were 0.15, 0.07, 0.22, 0.29, 2.70, and 4.53 (X 10(9) M-1) for Eq, E1, 17 beta-Eq, E2, T, and 5 alpha-DHT, respectively. These results indicate that the equine estrogens bind to SHBG and albumin in a manner similar to that of E1 and E2, and that the low MCR of EqS reported previously may be due to its binding to albumin.