Fishes possess more genes than other vertebrates, possibly because of a genome duplication event during the evolution of the teleost (ray-finned) fish lineage. To further explore this idea, we cloned five genes encoding phosphoinositide-specific phospholipase C-delta (PLC-delta), designated respectively PoPLC-deltas, from olive flounder (Paralichthys olivaceus), and we performed phylogenetic analysis and sequence comparison to compare our putative gene products (PoPLC-deltas) with the sequences of known human PLC isoforms. The deduced amino acid sequences shared high sequence identity with human PLC-delta1, -delta3, and -delta4 isozymes and exhibited similar primary structures. In phylogenetic analysis of PoPLC-deltas with PLC-deltas of five teleost fishes (zebrafish, stickleback, medaka, Tetraodon, and Takifugu), three tetrapods (human, chicken, and frog), and two tunicates (sea squirt and pacific sea squirt), whose putative sequences of PLC-delta are available in Ensembl genome browser, the result also indicated that the two paralogous genes corresponding to each PLC-delta isoform originated from fish-specific genome duplication prior to the divergence of teleost fish. Our analyses suggest that an ancestral PLC-delta gene underwent three rounds of genome duplication during the evolution of vertebrates, leading to the six genes of three PLC-delta isoforms in teleost fish.