We previously reported that MCF-7 cells were arrested in the G0/G1 phase of the cell cycle by agents known to block the activity of ATP-sensitive potassium channels (Woodfork et al., 1995, J. Cell Physiol. 162:163-171). The goal of our current study was to determine if MCF-7 cells undergo changes in membrane potential during the cell cycle that might be linked to changes in K permeability. The resting membrane potentials of unsynchronized MCF-7 cells during exponential growth phase were measured using sharp glass microelectrodes, and they ranged from -58.6 mV to -2.7 mV. The distribution of membrane potentials was best fitted by the sum of four Gaussian distributions with means of -9.0 mV, -17.4 mV, -24.6 mV, and -40.4 mV. These membrane potential groups were designated D (depolarized), ID (intermediate depolarized), IH (intermediate hyperpolarized), and H (hyperpolarized), respectively. The membrane potential was sensitive to the substitution of external K and Na but not Cl. The K:Na permeability ratio increased in proportion to the negativity of the membrane potential. MCF-7 cells pharmacologically arrested in G0/G1 phase were depolarized compared to control, with cells shifted from the H and IH groups to the D group. Tamoxifen-arrested cells chased from G0/G1 into S phase by the addition of mitogenic concentrations of 17 beta-estradiol were not depolarized, and these cells were shifted from the D group back to the IH and H groups. We conclude that MCF-7 cells hyperpolarize during passage through G0/G1 and into S phase, and this hyperpolarization probably results from an increase in the relative permeability of the plasma membrane to K.