In order to evaluate potential differences in the kinetics of peptide hormone-receptor interactions in hepatocytes of different species, we developed a simple procedure for the isolation of canine hepatocytes. Cells (obtained by collagenase perfusion of an extirpated dog liver lobe) were isolated with uniform high viability and yield. In addition, isolated dog hepatocytes tolerated incubation for at least 4 hr in defined medium with only a slight decrease in viability and with no change in the kinetics of [125I]iodoinsulin or [125I]iodoglucagon binding to cell-surface receptors. Comparisons of peptide hormone interactions with isolated dog and rat hepatocytes showed that (i) [125I]iodoglucagon associated with specific membrane receptors more rapidly than did [125I]iodoinsulin, for both rat and dog hepatocytes and at both 30 degrees C and 37 degrees C; (ii) the steady-state binding of [125I]iodoglucagon at 30 degrees C was greater than that of [125I]iodoinsulin in dog hepatocytes, but the reverse relationship held in rat hepatocytes; (iii) the rate of dissociation of [125I]iodoinsulin from hepatocytes of both species was enhanced by the presence of the unlabeled hormone, whereas the rate of dissociation of receptor-bound [125I]iodoglucagon was enhanced by the presence of unlabeled glucagon only in hepatocytes derived from the dog; and (iv) [125I]iodopancreatic polypeptide bound to neither rat nor dog hepatocytes, although the [125I]iodotyrosylated peptide bound to rat hepatocytes with an unusually high apparent dissociation constant. While confirming essential findings of pancreatic hormone binding to isolated hepatocytes, this comparison suggests that both qualitative and quantitative aspects of hormone-target cell interactions can show interspecies variability.