Insulin, a mitogen for cultured chick embryo fibroblasts (Temin, H.M. (1968) Cancer 3, 771-787), has been employed to characterize the effects of mitogen/cell membrane interactions as it relates to growth. The specific binding of 125I-insulin to substratum-attached cells is time- and temperature dependent and is optimum at a pH of 7.0. Fetal calf and chicken sera, somatomedin "A/C mixed," and desalanine or native porcine insulin compete with 125I-insulin for membrane-binding sites. Proinsulin, although competing less effectively than native insulin for binding, is more effective than desoctapeptide insulin. Unrelated polypeptide hormones do not compete for 125I-insulin binding. The lowest concentration of insulin at which specific binding is detected is 0.1 nM. Scatchard plot analysis of the binding data indicates that there are two types of binding sites in confluent cultures of fibroblasts: one of high affinity (K1 = 2 to 6 X 10(8) M-1) and low capacity, the other of low affinity (K2 = 0.8 to 3.0 X 10(7) M-1) and high capacity. Approximately 1.9 and 7.1 X 10(3) molecules of insulin are bound at each site, respectively. A 10-min incubation at 24 degrees of the fibroblasts with 10 mug/ml of trypsin causes a 2-fold stimulation of specific 125I-insulin binding and a similar 2-fold increase in insulin-stimulated 2-deoxy-D-glucose uptake and thymidine incorporation. Neuraminidase treatment also produces a 37% increase in specific 125I-insulin binding but treatment with alpha-chymotrypsin or phospholipase C are without significant effect. The results of this and additional experiments support the hypothesis that trypsin treatment of chick embryo fibroblasts leads to an unmasking of 125I-insulin binding sites. Serum starvation of fibroblasts for 12 or 24 h produces a 2.5- to 5-fold increase in specific 125I-insulin binding. This increase is the result of an increase in the number of hormone-binding sites from 9 X 10(3) to 6 X 10(4) per cell which are predominantly of the low affinity type. There is no change in the affinity constants. The presence of camptothecin, or cordycepin, or cycloheximide in the incubation medium completely blocks the increase in number of 125I-insulin-binding sites resulting from serum starvation. The addition of native insulin to the medium of serum-starved cultures also blocks this increase. The magnitude of insulin-stimulated 2-deoxy-D-glucose uptake and thymidine incorporation correlates with the levels of occupancy of the low affinity 125I-insulin-binding sites in untreated fibroblasts. In fibroblasts cultured in the absence of serum, the marked increase in insulin-stimulated 2-deoxy-D-glucose uptake and thymidine incorporation parallels the increase in number of mitogen receptors. The concentration of insulin that produces a half-maximum stimulation of thymidine incorporation is calculated to be 5 X 10(-8) M. At this concentration of insulin, 42% of the receptor sites are occupied.