Rat liver cytosolic proteins were photoaffinity labeled with the synthetic steroid [3H]methyltrienolone in order to identify and characterize hepatic proteins that may participate in the intracellular binding and transport of steroid hormones and other sterols. A male-specific and a female-specific sterol-binding protein (SBP) that migrated to the 4 S region of a sucrose gradient and had similar molecular weights (male-specific 34-kDa protein (SBP34), female-specific 31-kDa protein (SBP31] were thus identified. Experiments were undertaken to determine the biochemical basis for the sex-specific expression of these two proteins. In vivo hormonal manipulations established that the female-specific expression of SBP31 could, in part, be accounted for by the suppressive effects of androgen on SBP31 levels in male rats. In contrast, androgen stimulated expression of the male-specific SBP34, while estrogen and the estrogen-regulated continuous plasma growth hormone profile that is characteristic of adult female rats were suppressive toward this protein. Unlike several other androgen-dependent hepatic proteins, however, SBP34 did not require an intact pituitary for androgen-stimulated expression, nor was its expression stimulated by the intermittent pulses of plasma growth hormone that are characteristic of adult male rats. SBP34 and SBP31 were not induced but were suppressed to various extents by dexamethasone, phenobarbital, and clofibrate, drugs that are known to induce other hepatic proteins involved in steroid binding and metabolism. Competition experiments revealed that SBP31 has a relatively broad ligand specificity, with significant competition for [3H]methyltrienolone binding exhibited by bile acids (chenodeoxycholic acid and lithocholic acid) and a range of steroid hormones (progesterone, estradiol, testosterone, and 5 alpha-dihydrotestosterone) when present in the low micromolar range. No binding was detected with this protein toward cholesterol, triamcinolone acetonide, 5 alpha-androstan-3 alpha,17 beta-diol, cholic acid, and deoxycholic acid. In contrast, SBP34 exhibited greater binding specificity, with competition for [3H]methyltrienolone binding observed only with primary bile acids (cholic acid and chenodeoxycholic acid) and their metabolites (deoxycholic acid and lithocholic acid). On the basis of these binding specificities and the relatively high concentration of bile acids found in the liver, it is proposed that SBP31 and SBP34 function in the intracellular binding and/or transport of bile acids.