Transepithelial electrical potential difference (VT) was measured across the vestibular labyrinth of the inner ear in vitro by puncturing the epithelial wall of the utricle with a glass microelectrode. A region of nonsensory cells of the utricle was isolated from the sensory regions by introducing columns of liquid Sylgard 184. Under control conditions, the VT of this region was +7.5 +/- 0.3 mV (means +/- SE), lumen positive. This potential difference was rapidly reduced by either 1 mM ouabain, 10-100 microM bumetanide, 0.5-5.0 mM Ba (in the bathing solution), or cooling, but not by the disulfonic stilbene, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. Changes in VT due to reductions of Cl or Na or to increases of K in the bathing solution in exchange for presumably impermeant ions were observed in this region and were compared with those in a preparation in which the insulating seals were absent. The K-induced voltage change was significantly higher in the unblocked preparation, a finding consistent with a high K permeability of the sensory cells. The voltage change due to reduction of Cl was not inhibited by Cl channel blockers (9-anthracenecarboxylate and diphenylamine-2-carboxylate) in the bathing solution. These results represent the first direct demonstration that the nonsensory cells of the utricle produce a lumen-positive active-transport potential and characterize some of the properties of the cell membranes in terms of their pharmacological sensitivities and net voltage responses to changes in the bathing medium ions Na, K, and Cl.