Mono-(2-ethylhexyl) phthalate (MEHP) is known to cause germ cell apoptosis in the testis of rats as a consequence of Sertoli cell damage. In the present study, a single dose of 2,000 mg/kg body weight MEHP was orally administered to Fischer 344 rats and the molecular apoptosis pathway was surveyed. Histopathological examination, TUNEL assay and immunohistochemical analysis of cleaved caspase-3 were performed to assess the reproducibility of apoptosis. In addition, immunohistochemical analysis and RT-PCR to determine the parameters of the apoptosis pathway were performed. Histopathological examination showed that from 6 to 12 hours after MEHP treatment there was detachment of germ cells that had eosinophilic cytoplasm and pyknotic nuclei. A TUNEL assay detected a significant increase in TUNEL-positive cells at 12 hours after treatment. Immunohistochemistry showed some of the cytoplasm of germ cells was positive for cleaved caspase-3 at 12 hours after treatment. In immunohistochemistry of the Fas/Fas ligand (FasL) apoptosis pathway, the cytoplasm of germ cells was found to be occasionally positive for Fas at 12 hours after treatment, and the expression of the Fas gene in RT-PCR had significantly increased at 12 hours after treatment. RT-PCR of the mitochondrial apoptosis pathway revealed a significant increase in the expression of Bax and Bcl-2 genes in the treated group at 12 hours after treatment. These results strongly suggest that both the Fas/FasL and mitochondrial pathways are involved in germ cell apoptosis induced by MEHP.