Pepsinogen is a precursor of pepsin, a gastric specific protease belonging to the aspartic proteinase family. In teleosts, several species, such as zebrafish and puffer, have independently lost gastric glands. So whether puffer have pepsinogen gene or not is an interesting issue. A search of GSS database for pufferfish, Takifugu rubripes, revealed five different aspartic proteinase genes in its genome. One of them (pPep) has typical pepsinogen structure and belongs to the fish pepsinogen cluster by phylogenic analysis. The pPep antisense probe hybridized to the gastric glands of flounder stomach. Therefore, we concluded that pPep is a pufferfish pepsinogen. The pufferfish pepsinogen mRNA was not expressed in the digestive organs but specifically in the skin. We speculated that while Tetraodontiformes evolutionarily lost gastric glands, pufferfish pepsinogen acquired an alternative function to food digestion.