Microelectrodes and electron microprobe analysis were employed to study the effect of novobiocin on membrane potential and intracellular electrolyte concentrations in the frog skin epithelium. In both species investigated (Rana esculenta and Rana temporaria), novobiocin (1 mM, outer bath) caused a stimulation of transepithelial Na transport, a depolarization of apical membrane potential, a fall in the apical fractional resistance, and an increase in the intracellular Na concentration. The rise in the Na concentration was accompanied by an equivalent fall in the K concentration. All effects of novobiocin were fully reversible by subsequent application of amiloride. The depolarization as well as the Na increase suggests that the natriferic effect of novobiocin is due to a stimulation of the apical Na influx. Combining both measurements it was possible to calculate the effect of novobiocin on the Na permeability of the apical membrane directly. In Rana esculenta novobiocin increased the permeability from 4.5 to 23.2 nm/s. In Rana temporaria the increase was significantly smaller, from 8.7 to 16.9 nm/s. The transport rate as measured by the short-circuit current showed a non-linear dependence on the apical Na permeability. In the range of transport rates normally encountered, however, the current was a linear function of the Na permeability consistent with the view that the apical membrane is rate-limiting in transepithelial Na transport.