We have identified a novel polypeptide toxin (Lsp-1) from the venom of the spider Lycosa (LS). Its effect has been examined on the P-type calcium channels in Purkinje neurons, using whole-cell patch-clamp. This toxin (at saturating concentration 7 nM) produces prominent (four-fold) deceleration of the activation kinetics and partial (71 ± 6%) decrease of the amplitude of P-current without affecting either deactivation or inactivation kinetics. These effects are not use-dependent. They are partially reversible within a minute upon the wash-out of the toxin. Intracellular perfusion of Purkinje neurons with 100 μM of GDP or 2 μM of GTPγS, as well as strong depolarising pre-pulses (+100 mV), do not eliminate the action of Lsp-1 on P-channels indicating that down-modulation via guanine nucleotide-binding proteins (G-proteins) is not involved in the observed phenomenon. In view of extremely high functional significance of P-channels, the toxin can be suggested as a useful pharmacological tool.