The plasmamembrane potential (delta psi p) of murine erythroleukemia (MEL) cells has been determined by measuring the distribution of the lipophilic cation tetraphenylphosphonium (TPP+) across the plasmamembrane. TPP+ accumulation within the cells (experimental accumulation ratio, AR exp) was measured by adding 2 microM TPP+ directly to the culture flasks, avoiding any other perturbation of the experimental system. The mitochondrial contribution to AR exp, evaluated by adding carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or 2,4-dinitrophenol (DNP), was apparently negligible in standard cultures, AR exp being substantially the same in either the absence or presence of these uncouplers. However, the addition of oligomycin produced a strong AR exp enhancement, which was abolished by FCCP, suggesting that MEL cell mitochondria are in state 3. The aspecific TPP+ binding was estimated by a new mathematical approach worked out to fit AR exp values measured in the presence of valinomycin at various extracellular K+ concentrations and plotted against the ratio of intracellular to extracellular K+ concentration ([K+]i/[K+]e). This binding was found to be close to zero in MEL cells. By applying the Nernst equation directly to AR exp values, delta psi p of these cells was then measured; this potential varying from -65 mV to -16 mV (inside negative) is inversely related to the cell density on the culture surface on which the cells sediment (cells/cm2; CD). The dependence of delta psi p on CD is practically eliminated by valinomycin and appears to be related to a cell interaction with the culture surface of the flasks, suggesting that in the immediate environment of MEL cells one or more factors are produced that modulate the K+ plasma membrane permeability (PK).