Summary The effects of different periods of incubation (8 min vs 20 min) on insulin binding kinetics were examined in a H35 hepatoma cell line. Scatchard plots from cells incubated for 8 min were linear (r=0.987±0.006), in contrast to curvilinear Scatchard plots from cells incubated for 20 min. Hill plots showed a slope of 1.006±0.024 for the 8 min incubation, whereas the slope was 0.827±0.0026 (p<0.0005) for the 20 min incubation. TCA precipitation of the medium showed minimal insulin degradation products at 8 min with a significant increase at 20 min (1.38 ± 0.11% vs. 3.06 ± 0.37%, p<0.005). Internalized insulin was also significantly increased at 20 min as compared to 8 min incubation (48.9 ± 5.6% vs. 32.4 ± 3.0%, p<0.0005) These data indicate that after 8 min of incubation no appreciable cooperativity of insulin binding was present, while negative cooperativity was present after 20 min of incubation. As significantly more insulin degradation has taken place after prolonged incubation these data support the hypothesis that insulin degradation leads to negative cooperativity of insulin receptors.