A cDNA encoding a novel phospholipase A(2) (PLA(2)) inhibitor (PLI) was isolated from a Protobothrops flavoviridis snake (Tokunoshima island, Japan) liver cDNA library. This cDNA encoded a signal peptide of 19 amino acids followed by a mature protein of 181 amino acids. Its N-terminal amino acid sequence was completely in accord with that of a PLI, named PLI-II, previously found in P. flavoviridis serum. PLI-II showed a high similarity in sequence to the B subtype of gammaPLI, denoted gammaPLI-B, isolated from Agkistrodon blomhoffii siniticus serum. Thus, PLI-II is P. flavoviridis serum gammaPLI-B. Since PLI-I, previously isolated from P. flavoviridis serum, can be assigned as gammaPLI-A, P. flavoviridis serum contains both A and B subtypes of gammaPLI. Phylogenetic analysis of gammaPLIs from the sera of various kinds of snakes, Elapinae, Colubrinae, Laticaudinae, Acanthophiinae, Crotalinae, and Pythonidae, based on the amino acid sequences revealed that A and B subtypes of gammaPLIs are clearly separated from each other. It was also found that phylogenetic topologies of gammaPLIs are in good agreement with speciation processes of snakes. The BLAST search followed by analyses with particular Internet search engines of proteins with Cys/loop frameworks similar to those of PLI-II and PLI-I revealed that gammaPLI-Bs, including PLI-II and PLI-II-like proteins from mammalian sources, form a novel PLI-II family which possesses the common Cys/loop frameworks in the anterior and posterior three-finger motifs in the molecules. Several lines of evidence suggest that PLI-II is evolutionarily ancestral to PLI-I.