In order to test whether prostaglandins (PGs) function as sex pheromones in Hynobius leechii, a salamander that externally fertilizes its eggs, we conducted electro-olfactogram (EOG) studies with 19 PGs, liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses of female and male holding waters, and behavioral tests on selected PGs. Of the 19 PGs tested, only three induced strong EOG responses from both males and ovulated females: 15-epi-prostaglandin F2alpha (15(R)-PGF2alpha), 15-keto-prostaglandin F2alpha (15K-PGF2alpha), and 13,14-dihydro-15-keto-prostaglandin F2alpha (13,14-dh-15K-PGF2alpha). In the LC-MS/MS studies, samples of holding water from ovulated females contained higher concentrations of 15(R)-PGF2alpha, PGF2alpha, and 13,14-dh-15K-PGF2alpha than those from males or oviposited females. In the behavioral tests, only 15(R)-PGF2alpha and ovulated female holding water induced significant reproductive behavior from male salamanders. These results suggest that F-series prostaglandins function as sex pheromones in amphibians.