The net influx of L-arginine (JARG) was employed as an indicator of the membrane potential in human fibroblasts. Cell depolarization, obtained by increasing [K+]out, decreased both JARG and the net influx of the lipid soluble cation tetraphenylphosphonium (JTPP), a probe of membrane potential. JTPP, but not JARG, was influenced by the mitochondrial potential and exhibited a component dependent on intracellular and/or extracellular binding. JARG was sensitive to changes in the membrane potential induced by Na+-dependent transport of L-proline or by the activity of Na+-K+-ATPase. In the presence of 50 microM valinomycin, JARG was markedly influenced by the distribution ratio of K+ in a range of [K+]out from 1.5 to 100 mM. In this range of [K+]out, membrane potential (Em) varied from -90 to -23 mV, and calibration of JARG vs. the membrane potential yielded a linear relationship. These results indicate the following: 1) that the net influx of TPP+ is not a reliable indicator of membrane potential in cultured human fibroblasts; 2) that in the same cells the net influx of L-arginine can be employed as an index of membrane potential; 3) that in a range of Em from -23 to -90 mV the activity of system y+ (the membrane agency devoted to L-arginine transport in cultured human fibroblasts) exhibits no saturation of potential-dependent activation of transport.