Bovine non-pigmented ciliary epithelial cells (NPE) have been isolated by a technique of selective adhesion to tissue culture plastic. NPE cells in primary culture proliferated and maintained epithelial-like morphology for about 4 weeks in tissue culture medium containing 10% fetal calf serum. If grown for longer than 4 weeks in serum-containing medium, cells changed their morphology and became elongated and spindle-shaped. Membrane potentials were measured using conventional microelectrodes. In NPE cells of epithelial-like shape, replacing extracellular Na⁺ induced a transient hyperpolarization of the membrane potential, while in elongated cells of spindle-shaped morphology an immediate depolarization was observed. We therefore only used epithelial-like NPE for further experiments. In these cells the mean membrane potential was -40.3±0.5 mV (n=36). Relative K⁺ conductance was increased by extracellular alkalinization. Removing extracellular K⁺ led to a depolarization and readdition of K⁺ to K⁺ depleted cells resulted in a hyperpolarization. Both voltage responses were sensitive to ouabain, indicating that Na⁺/K⁺ ATPase is inhibited by K⁺ replacement, and that there is overshoot-activation of the pump when K⁺ is readded. Extracellular Cl⁻ replacement led to a DIDS sensitive, transient depolarization, which is compatible with a stil-bene-sensitive Cl⁻-conductance. Removing HCO₃⁻ led to a Na⁺ dependent and DIDS-sensitive depolarization. However, the electrical response on replacement of extracellular Na⁺ was not influenced by DIDS or the extracellular HCO₃⁻-concentration.