Erythrocyte lysate increases intracellular Ca<sup>2+</sup> ([Ca<sup>2+</sup>]<sub>i</sub>)and contracts cerebral arteries in vitro and has been suggested to be the cause for cerebral vasospasm. We investigated the effect of hemolysate on L-type Ca<sup>2+</sup> channels directly by using patch clamp techniques in freshly isolated single smooth muscle cells from rat basilar artery. Patch clamp studies revealed a whole-cell current which resembles the L-type Ca<sup>2+</sup> current reported by others. Hemolysate reduced the amplitude of the L-type Ca<sup>2+</sup> channel current. The effect of hemolysate was reversible by washout and repeatable. Hemolysate was separated into two fractions by using filter membranes. The fraction > 1 kDa which contains oxyhemoglobin and other proteins mimicked the effect of hemolysate, while the fraction < 1 kDa and ATP were without effect. We conclude that hemolysate does not increase [Ca<sup>2+</sup>]<sub>i</sub> by activation of L-type Ca<sup>2+</sup> channel.