Transmembrane potentials of rat hepatocytes in primary monolayer culture on collagen gels were measured with glass microelectrodes. Potentials for cells in culture for 23-30 h comprised two populations. The mean +/- SD for a population of stable low potentials was -9.7 +/- 2.0 mV (n = 93). This was compared with -23.6 +/- 9.4 mV (n = 42), the mean value for stable potentials that followed spontaneous increases in the low potentials, 0.5-2.0 min after the impalement. The estimated input resistance increased during these spontaneous hyperpolarizations. In some cells, after 48 h in culture, the transmembrane potential oscillated rhythmically, with an amplitude of 25 mV and a period of 7 min. Suffusing the cells with 120 mM potassium chloride decreased the potential and eliminated the oscillations. The stable high potentials were considered more accurate estimates of the hepatocyte transmembrane potential, based on comparison with values for intact liver. Low potentials may have resulted from current leaking through an electrode shunt resistance, followed by an increase in potential as the membrane "sealed" the shunt pathway. However, these events may also reflect cells capable of two stable transmembrane potentials.