As a function of buffer pH, [125I]-insulin binding to rat mammary cells, rat adipocytes, or membranes prepared therefrom, at 4 degrees or 20 degrees C, showed 2 peaks in different buffers. Specific insulin binding at the pH 7.7. peak (100 +/- 11%) was lower than at pH 8.8 (140 +/- 17%) with no change in nonspecific binding. Although insulin stimulation of glucose uptake into fat cells was highest at pH 7.5, this response was also seen at pH 8.6. Scatchard affinity profiles, or in the kinetics of dissociation. Insulin degradation (< 10%) and binding to insulin antibody were similar over the pH range of 7 to 9.