Kinase activities were previously proposed to be central to germ cell apoptosis induced by ethylene glycol monomethyl ether (EGME) and its active metabolite methoxyacetic acid (MAA). We evaluated the role of tyrosine kinase pp60(c-src) in control and EGME-treated adult rat testis in vivo, as well as in vitro using cultured adult rat seminiferous tubules treated with MAA. In normal testicular tissue, immunoreactivity of Src was mostly detected in Sertoli cell cytoplasm and reached the maximum level around the lumen at spermiation. Src localization was confirmed by immunostaining of cocultures of Sertoli and germ cells and was further confirmed by electron microscopic observation that immunoreactivity was predominant in Sertoli cell cytoplasm as well as occasionally at the Sertoli/germ cell junctions. A single dose of 200 mg/kg EGME induced an increase of Src immunoexpression in both epithelium and interstitium in rat testis. Eight hours after treatment, an intensive immunostaining of Src began to be observed specifically in the cytoplasm of the dying spermatocytes. The apoptotic changes were replicated by exposure of 5 mM MAA in the adult rat seminiferous tubule culture model. Furthermore, spermatocyte degeneration was significantly prevented by cotreatment with 0.1 microM geldanamycin, 10 microM herbimycin A, or 10 microM PP2, which are inhibitors of Src activity. These data collectively suggest that pp60(c-src) mediates Sertoli-germ cell interaction in physiological events, and may play an important role in EGME/MAA-induced germ cell apoptosis.