Substitution of acetate for external Cl produced a large persistent increase in the resting membrane potential (R.M.P.) of frog ventricle and a somewhat steeper relation between membrane potential (M.P.) and [K]o (external K concentration). An increased K conductance or reduced permeability to other ions could account for most of these results, but not for hyperpolarizations as great as −110 mV. Potentials of this size suggested a contribution from an active electrogenic transport system, but they were unaffected by several treatments including exposure to ouabain (10−7 M − 5 × 10−6 M), dinitrophenol (10−6 M, 10−5 M) or 30 mM tetraethylammonium.Acetate caused a prolongation of the action potential (A.P.) and a change in its configuration. Acetate also enhanced twitch tension and increased the rate of tension development. Similar changes are produced by removal of [K]o. The effects of both acetate and K removal on A.P. configuration were prevented by a reduced rate of stimulation.When acetate-induced hyperpolarization was reversed by raising [K]o to 10–15 mM, the configuration of the A.P. resembled that of controls and twitch tension did not increase. Thus, acetate-induced changes in the shape of the A.P. and in twitch tension appeared to be secondary to the increase in R.M.P. However, the relationship does not seem to be direct because these changes were temporary, whereas hyperpolarization was persistent.The character of the acetate-induced changes in A.P. configuration, and the dependence on stimulation rate and [Ca]o (external Ca concentration), suggested a raised [Ca]i (internal Ca concentration) and a possible increase in Ca influx. However, addition of Mn to the acetate solution did not prevent initial acetate-induced changes in the shape of the A.P. plateau and in twitch tension. Also in the absence of [Ca]o, disappearance of twitch tension was slowed by acetate. But acetate decreased the contracture tension produced in response to either increased [K]o or Na removal. Acetate may cause a redistribution of Ca within the cell.