We report analyses of the effect of anti-Fab antibodies on plasma membrane potential of mouse B lymphocytes. Results indicate that divalent fragments of anti-Fab antibodies mitogenic for B cells stimulate membrane depolarization detectable by cytofluorometric analysis of 3,3'-dipentyloxacarbocyanine iodide-stained cells. Depolarization is detectable within 5 min of exposure to ligand and maximal within 1 h of exposure when greater than or equal to 80% of splenic B cells exhibit decreased membrane potential. The ineffectiveness of monovalent Fab antibody fragments in inducing this event suggests that receptor immunoglobulin cross-linking is essential. Frequencies of cells induced to enter cell cycle, as assessed by acridine orange cell cycle analysis, are equal to those induced to depolarize by lipopolysaccharide plus dextran sulfate or anti-Fab, which suggests a relationship between these events. However, membrane depolarization is itself an insufficient signal to promote subsequent thymidine uptake, as evidenced by the fact that doses of anti-Fab that are suboptimal for thymidine uptake induce maximal depolarization. These results suggest that cross-linking of surface immunoglobulin on B cells may provide an initial signal for activation but is itself insufficient to drive B cell proliferation.