The in vitro effects of two biguanides (phenformin and metformin) and four sulfonylureas (tolbutamide, glyburide, gliclazide, and glisolamide) on insulin binding to its receptors were studied in four cultured cell lines: human skin fibroblasts, IM-9 lymphoblasts, MCF-7 human mammary carcinoma, and H35 rat hepatoma. After a 24-h preincubation with maximal stimulatory concentrations of phenformin, specific [125I] insulin binding to its receptors in the four different cell lines were increased over control by 67.2 +/ 17.0%, 101.3 +/- 11.5%, 65.1 +/- 8.0%, and 44.0 +/- 12.1%, respectively (mean +/- SE). Phenformin was effective in IM-9 cells that were down-regulated by unlabeled insulin, and the effect of phenformin on insulin binding was not affected by inhibition of protein synthesis with cycloheximide. In concert with this observation. Scatchard plots indicated that phenformin increased the insulin receptor's affinity rather than the number of insulin-binding sites on IM-9 cells. Metformin was also effective in significantly enhancing insulin binding in both IM-9 and MCF-7 cells. In contrast to the effects of biguanides, none of the four sulfonylureas tested had any significant influence on insulin binding to any of the four cell lines. These agents were also ineffective in IM-9 cells that were down-regulated by insulin. Therefore, these studies suggest that: 1) in vitro, biguanides enhance insulin binding to its receptors in a variety of cell types; 2) this effect of biguanides doesn't depend on new receptor synthesis; it is a result of changes in the affinity of the insulin receptor; and 3) in contrast to the biguanides, the sulfonylureas do not have a major direct effect on insulin binding to its receptors in most cell types.