Neuroblastoma-glioma hybrid cells (NG108-15) in suspension accumulate the permeant lipophilic cation [3H]tetraphenylphosphonium (TPP+) against a concentration gradient. The steady-state level of TPP+ accumulation is about twice as great in physiological media of low K+ concentration (i.e., 5 mM K+/135 mM Na+) than in a medium of high K+ concentration (i.e., 121 mM K+/13.5 mM Na+). The latter manipulation depolarizes the NG108-15 plasma membrane and indicates that the resting membrane potential (ΔΨ) is due primarily to a K+ diffusion gradient (Kin+ → Kout+). TPP+ accumulation is time and temperature dependent, achieving a steady state in 15-20 min at 37°C, and is a linear function of cell number and TPP+ concentration (i.e., the concentration gradient is constant). The difference in TPP+ accumulation in low and high K+ media under various conditions has been used to calculate mean (±SD) ΔΨ values of -56 ± 3, -63 ± 4, and -66 ± 5 mV at 26, 33, and 37°C, respectively. Importantly, these values are virtually identical to those obtained by direct electrophysiological measurements made under the same conditions. TPP+ accumulation is abolished by the protonophore carbonylcyanide-m-chlorophenylhydrazone, whereas the neurotoxic alkaloid veratridine diminishes uptake to the same level as that observed in high K+ media. In addition, the effect of veratridine is dependent upon the presence of external Na+ and is blocked by tetrodotoxin. The steady-state level of TPP+ accumulation is enhanced by monensin, indicating that this ionophore induces hyperpolarization under appropriate conditions. Finally, ouabain has essentially no effect on the steady-state level of TPP+ accumulation in short-term experiments, suggesting that Na+,K+-ATPase activity makes little contribution to the resting potential in these cells. Because many of these observations are corroborated by intracellular recording techniques, it is concluded that TPP+ distribution measurements can provide a biochemical method for determining membrane potentials in populations of cultured neuronal cells.