We tested the hypothesis that cyclic changes in membrane potential (Em) underlie spontaneous vasomotion in cheek pouch arterioles of anesthetized hamsters. Diameter oscillations (∼3 min–1) were preceded (∼3 s) by oscillations in Em of smooth muscle cells (SMC) and endothelial cells (EC). Oscillations in Em were resolved into six phases: (1) a period (6 ± 2 s) at the most negative Em observed during vasomotion (–46 ± 2 mV) correlating (r = 0.87, p < 0.01) with time (8 ± 2 s) at the largest diameter observed during vasomotion (41 ± 2 µm); (2) a slow depolarization (1.8 ± 0.2 mV s–1) with no diameter change; (3) a fast (9.1 ± 0.8 mV s–1) depolarization (to –28 ± 2 mV) and constriction; (4) a transient partial repolarization (3–4 mV); (5) a sustained (5 ± 1 s) depolarization (–28 ± 2 mV) correlating (r = 0.78, p < 0.01) with time (3 ± 1 s) at the smallest diameter (27 ± 2 µm) during vasomotion; (6) a slow repolarization (2.5 ± 0.2 mV s–1) and relaxation. The absolute change in Em correlated (r = 0.60, p < 0.01) with the most negative Em. Sodium nitroprusside or nifedipine caused sustained hyperpolarization and dilation, whereas tetraethylammonium or elevated PO2 caused sustained depolarization and constriction. We suggest that vasomotion in vivo reflects spontaneous, cyclic changes in Em of SMC and EC corresponding with cation fluxes across plasma membranes.