Rat hepatocytes in primary monolayer culture demonstrated two stable states of transmembrane potential (Em). Low potentials ranging from -10 to -15 mV followed impalements with glass microelectrodes, and in some cells low potentials increased spontaneously or in response to a train of intermittent current (5 nA) to stable potentials of -50 mV, which were comparable to hepatocyte Em in vivo. Adding insulin at 20 mU/ml depolarized the stable higher Em 22.4 +/- 2.6 mV (n = 6) over an 11-min interval, and input resistance increased 21.4 +/- 4.7 M omega (n = 6) during the depolarization. The insulin effect was dose dependent, because adding insulin at 0.2 mU/ml depolarized the stable high Em 5.0 +/- 1.5 mV (n = 3) and increased input resistance 6.3 +/- 1.8 M omega (n = 3). In one experiment the Em repolarized 32 min after insulin was washed out. Adding metabolic inhibitors KCN (1 mM) and 2,4-dinitrophenol (10 and 1 mM) and increasing external potassium (60 mM, with external sodium reduced equivalently) also depolarized Em, but they did not increase input resistance. Thus insulin depolarized hepatocytes from a stable high Em, which is equivalent to the Em of liver in vivo, to a stable low Em, which occurs in hepatocytes in primary monolayer culture. This hormone action is consistent with changes in membrane ion conductance, and it further demonstrates that these cells can sustain two stable states of Em.