Normal rat kidney cells (NRK) infected with the temperature-sensitive (ts) transformation mutant of Moloney murine sarcoma virus yielded a clone of cells, 6m2, that exhibited a transformed morphology at 33 degrees C and a normal morphology at 39 degrees C. Transmembrane potential (Em) was measured fluorometrically using a cyanine dye diS-C3-(5). Fluorescence was inversely correlated with Em. Cells at 33 degrees C had lower Em. Em changes were recorded within 15 minutes of temperature shift from 33 degrees C to 39 degrees C in both directions, increasing in the 33 degrees C to 39 degrees C direction and decreasing in the 39 degrees C to 33 degrees C direction. Uninfected NRK cells when shifted under the same condition exhibited small fluorescence changes in the 33 degrees C to 39 degrees C direction. Shifting from 39 degrees C to 33 degrees C resulted in Em changes similar to those in 6m2 cells. Also studied was a cell line infected with a spontaneous revertant of the ts mutant, designated 54-5A4; it was transformed at both temperatures. Shifting from 33 degrees C to 39 degrees C in both directions yielded small changes. Transmembrane potential changes in 6m2 cells precede other transformation-specific changes that occur after a temperature shift.