The receptor binding characteristics and biologic potency of biosynthetic human proinsulin (rDNA) were determined in isolated rat adipocytes and compared with those of insulin. In competition with ¹²⁵I(A14)-iodoinsulin for binding to adipocyte receptors at 15°C, proinsulin showed a 100-fold lower affinity for binding than did insulin. A proinsulin concentration of 3.2 ± 0.8 × 10⁻⁷ M was required for 50% inhibition of tracer binding as compared with a concentration of 1.7 ± 0.3 × 10⁻⁹ M for insulin. These results were confirmed in direct competition studies using proinsulin and ¹²⁵I-iodoproinsulin. A similar 100-fold difference was also observed in competitive binding experiments conducted at 37°C. The biologic potency of human proinsulin was evaluated by its ability to stimulate glucose incorporation into total fat cell lipid and also by its antilipolytic activity. Glucose incorporation into lipid was half-maximal at a proinsulin concentration of 1.5 ± 0.4 × 10⁻⁸ M, whereas the same response was observed at an insulin concentration of 5.2 ± 1 × 10⁻¹¹ M. Proinsulin also demonstrated an antilipolytic potency that was <1% that of insulin. The time course over which insulin and proinsulin stimulated glucose incorporation into lipid was the same, as was the time course over which the stimulation dissipated after removal of the hormones. No synergism of insulin and proinsulin stimulation of lipogenesis was observed when fat cells were incubated with submaximal concentrations of the two hormones. It is concluded that biosynthetic human proinsulin has approximately 1% the receptor binding affinity and biologic potency of insulin in the isolated rat adipocyte system.