Specific binding of 125I-insulin was found in cultured human endothelial cells obtained from human umbilical veins. The binding reaction was rapid and reversible, demonstrated receptor site-site interactions of the negatively cooperative type, and was dependent on the temperature, pH and duration of incubation. At 21 degrees C, steady-state conditions of binding occurred in 90 minutes, the pH optimum was 7.8 and less than 10% of the labeled hormone was degraded. Binding of tracer amounts of 125I-insulin was inhibited by concentrations of unlabeled insulin as low as 0.2 ng/ml and 50% inhibition was obtained at 2-5 ng/ml of unlabeled insulin. Unlabeled porcine insulin, porcine proinsulin and desoctapeptide insulin inhibited the binding of 125I-porcine insulin in direct proportion to their biological potencies, whereas to the cells was inhibited by antibodies against insulin receptors. We conclude that human endothelial cells possess specific receptors for insulin whose physio-chemical properties are similar to those of insulin receptors in other tissues.