Inhibition of insulin degradation by the use of chloroquine and bacitracin leads to a straight line in the Scatchard plot after correction for internalized insulin (r = 0.978 +/- 0.011). Analysis according to a model with two groups of heterogeneous binding sites showed a significant reduction in the total number of binding sites (p less than 0.01) in the chloroquine-bacitracin treated cells, with a disappearance of the low affinity binding sites and an increase in the assessment of the number of high affinity binding sites (p less than 0.002). The assessment of the high affinity association constant showed a decrease in the chloroquine-bacitracin treated cells (p less than 0.05). Analysis according to a model with a homogeneous group of binding sites with negative cooperativity also showed a decrease in the assessment of the number of binding sites (p less than 0.05) with a significant reduction in the Ke/Kf ratio from 4.5 +/- 1.5 to 1.16 +/- 0.22 (p less than 0.002). These results indicate that inhibition of insulin degradation leads to disappearance of negative cooperativity; the data are inconsistent with the two site model.