The effect of 1 mmol/l H2O2 was studied on the membrane potential and [Ca2+]i with microelectrodes and the fura-2 technique, respectively. H2O2 induced a biphasic increase in [Ca2+]i with a fast transient peak and a subsequent plateau. H2O2 also led to a biphasic hyperpolarization of the cells with a similar time course. This was followed by a slight depolarization after wash-out of H2O2. External Ca2+ free solutions and treatment with the Ca2+ ionophore A23187 (1 mumol/l) abolished the effect of H2O2 on [Ca2+]i and almost entirely reduced the effect on the membrane potential. Phenylephrine (10 mumol/l) or A23187 also induced very similar biphasic hyperpolarizations of the membrane as H2O2 which were fully reversible after wash-out. It is concluded that H2O2 hyperpolarizes the membrane by opening of Ca2+ dependent K+ channels.