Binding sites of isolated rat pancreatic islets have been shown to interact with insulin. Employing various species-insulins, insulin analogues and substances not being structurally related to insulin, structure-specificity as well as pH- and temperature-dependence of insulin binding to rat pancreatic islets have been studied. Rat insulin displaced 125 I-insulin from its binding sites in the same concentration-dependent manner as pork insulin did, whereas the insulin analogue des-(phe-val-asp)B1-3-p-glu B4-insulin was less effective. Pork C-peptide hardly competed for binding and pork proinsulin did not compete at all. Both the species' insulins inhibited glucose (16.7 mM)-induced insulin secretion. The inhibitory effect was less when des-(phe-val-asp)B1-3-p-glu B4-insulin was employed and no inhibition of insulin secretion was observed by the use of pork C-peptide or proinsulin. Glucagon and somatostatin did not affect insulin binding. pH optimum of insulin binding appears to be in the range between 7.0 and 8.0. Binding was augmented with increasing temperature up to 37 degrees C. It is concluded that rat pancreatic islets possess insulin because binding and biological potency of substances related to insulin were in harmony. Moreover pH- and temperature-optimum of insulin binding are in a physiological range.