Binding of insulin and Concanavalin A to primary cell cultures of the R3230AC rat mammary adenocarcinoma was studied as a function of time in culture. As the culture became confluent, the amount of insulin binding per cell increased with culture time and reached a plateau, whereas the binding of Con A to surface glycoproteins decreased to 50% of the initial value. Exposure of confluent cultures to insulin at 37 C resulted in down-regulation of the cell surface insulin receptors. The decrease in insulin binding was related to the ambient insulin concentration and the decreased numbers of receptors per cell with no apparent alteration in their affinity. The maximal decrease in receptor number was 60-70%. Cell cultures degraded significant amounts of insulin at 37 C, but the addition of bacitracin to the culture medium decreased the amount of degradation and increased the extent of down-regulation at each insulin concentration. Porcine proinsulin was less effective than insulin in competing with 125I-labeled insulin and inducing receptor down-regulation. Down-regulation of insulin receptors did not require protein synthesis. The rate of insulin-induced receptor loss was much faster than the decrease in insulin binding due to inhibition of protein synthesis by cyclohexamide. The estimated half-life of the insulin receptor was 10.5 h. Down-regulation of insulin receptors was reversible; regeneration of receptors to 50% of control levels occurred approximately 9.6 h after the removal of insulin and required protein synthesis. These results indicate that these mammary tumor cells retain the ability to regulate their insulin receptors.