The resting potential (Em) of freshly isolated rabbit corneal endothelium was measured at room temperature (22 degrees C) and at 34 degrees C. Due to the wide range of values reported in the literature and the difficulty in obtaining long-term measurements using microelectrodes in these cells, a current-clamp technique was employed using whole cell patch-clamp electrodes. The electrodes contained a K+ methanesulfonate-based intracellular solution, and a NaCl/HCO3- Ringer's solution was used extracellularly. Three preparations of endothelium were examined: single dissociated cells, the isolated monolayer (stripped from the stroma with Descemet's membrane), and the intact isolated cornea. The perforated-patch technique, with amphotericin B in the electrode, was also used with the intact-cornea preparation at 34 degrees C. The mean Em values for the combined preparations at 22 degrees C and 34 degrees C were -35.3 mV and -55.0 mV, respectively; those for the intact-cornea preparation were -34.4 mV and -61.6 mV (at 22 degrees C and 34 degrees C, respectively). The isolated monolayer preparation showed a small but significant depolarization at both temperatures. These results demonstrate temperature dependence for Em in the corneal endothelium and show that more extensively dissected preparations have similar although not identical Ems to those of the intact cornea.