Differentiating (3T3-L1) and nondifferentiating (3T3-C2) fibroblastic cell lines possess two classes of insulin receptors, high affinity (KD = 1.0 to 3.7 X 10(-9) M) and low affinity (KD = 2.0 to 3.6 X 10(-8) M). Confluent cultures of 3T3-L1 cells induced to differentiate by insulin (1.74 x 10(-6) M) or indomethacin (1.25 x 10(-4) M) exhibit a 3-fold increase in the number of high affinity and low affinity receptors per cell or a 1.5- to 1.8-fold increase in the number of receptors per micron2 of surface area. In contrast, nondifferentiating 3T3-C2 cells treated with insulin or indomethacin lose almost completely the high affinity insulin receptors while retaining the same levels of low affinity receptors. The loss of high affinity receptors of the 3T3-C2 cells is accompanied by the disappearance of the stimulatory effect of insulin on the production of CO2 from glucose and on the uptake of aminoisobutyrate. The levels of high affinity insulin receptors appear to be regulated by different mechanisms in the differentiating (3T3-L1) and nondifferentiating (3T3-C2) cell lines. The mode of this regulation may have a bearing on the ability of a particular cell line to differentiate.