For the first time the venom of recently established viper species Vipera nikolskii was fractionated and two heterodimeric phospholipases A(2) (HDP-1 and HDP-2) were isolated. Isolation of HDP-1 and HDP-2 is the first indication of the presence of two heterodimeric phospholipases A(2) in the venom of one viper species. When tested on the frog neuromuscular junction, isolated proteins affected neuromuscular transmission acting presynaptically. Using RP-HPLC, each heterodimer was separated into two monomeric subunits: basic phospholipase A(2) (HDP-1P and HDP-2P) and acidic component without enzymatic activity (HDP-In). The complete primary structures of subunits were deduced from corresponding sequences of cDNAs. The determined amino acid sequences were homologous to those of vipoxin from Vipera ammodytes and vaspin from Vipera aspis. Similar proteins were not found earlier in the well-studied venom of Vipera berus, the species from which V. nikolskii was recently separated. Our finding supports at the biochemical level the correctness of the establishment of V. nikolskii as an independent species. The finding of similar proteins (HDPs and vipoxin) in geographically remote species (V. nikolskii and V. ammodytes) corroborates the hypothesis about the pre-existence of genes encoding these proteins in all true viper species and their expression under certain conditions.