The effect of disulfide reduction on the binding of [125I]rat GH (rGH) to rat liver plasma membranes and hepatocytes was studied to determine the role of disulfide bonds in the binding of GH to its receptor. The total amount of [125I] rGH bound to the liver receptors increased severalfold in the presence of dithiothreitol and mercaptoethanol. The nonspecific binding also increased at higher concentrations of the reductant, but the amount specifically bound was still greater in the presence of disulfide reductant. In contrast, the disulfide reductant inhibited [125I] human GH (hGH) binding and enhanced its displacement from hypophysectomized female rat hepatocytes. This was similar to the effect of reductants on [125I]hGH binding to normal female rat hepatocytes. The effect of the disulfide reductants on [125I]rGH binding could be prevented or reversed by the simultaneous or subsequent addition of an oxidizing agent such as NAD or oxidized glutathione. Sulfhydryl-reactive agents such as iodoacetamide prevented additional binding of [125I]rGH when added at 30 min of the incubation. The additional [125I] rGH bound in the presence of disulfide reductant was displaceable by excess unlabeled rGH. Both rGH and hGH exhibited similar degrees of disulfide reduction in the presence of mercaptoethanol. The disulfide reductant produced effects on binding at concentrations that resulted in less than 10% reduction of the GH disulfides. We conclude that: 1) the disulfides and sulfhydryls of the hepatocyte membrane are intimately involved in the binding of GH to hepatic receptors; 2) the locus of the disulfides and sulfhydryls may be in the subunit structure of the membrane receptor, but this will require verification using soluble receptors; and 3) the effect of disulfide reducing agents reveals basic differences in the mechanism of binding of rGH and hGH to somatotropic hormone receptors on the hepatocytes.