We have investigated the effect of the venom of a Chilean tarantula, Phrixotrichus spatulatus, on cell contraction, intracellular [Ca2+] ([Ca2+]i), and the L-type Ca2+ current (ICa,L) in cells isolated from the ventricles of guinea pig hearts. Whole-cell voltage clamp techniques were used to monitor ICa,L. The action potential was recorded using whole cell current clamp. [Ca2+]i was monitored using the fluorescent indicator indo-1. The venom of P. spatulatus decreased ICa,L in a dilution-dependent manner, with half-maximal inhibition at a dilution of 1.1/10(4) (v/v). At a dilution of 1/10(4), this inhibition occurred at all potentials so that the current-voltage relationship of ICa,L was depressed. However, inhibition of ICa,L by the venom was relieved by positive potentials, the venom being less effective following pulses to positive potentials. The venom reduced the duration of the action potential (at 50% repolarization) by between 26 and 43%. The venom also decreased the amplitude of the [Ca2+]i transient and cell contraction. It is concluded that the venom of P. spatulatus is a potent, voltage-dependent inhibitor of ICa,L; this inhibition of ICa,L may account for the effects of the venom on action potential duration, [Ca2+]i, and contraction.