The distinction between seminoma and embryonal carcinoma based on morphology alone can sometimes be problematic, requiring the use of immunohistochemistry to facilitate diagnosis. D2-40 is a monoclonal antibody that reacts with an oncofetal antigen expressed by fetal germ cells and testicular germ cell tumors. The diagnostic value of D2-40 immunohistochemistry in distinguishing seminoma from embryonal carcinoma has not been determined. D2-40 immunoreactivity was evaluated in a series of testicular germ cell tumors and compared with that of KIT (CD117) and CD30, to assess the relative utility of this marker in discriminating between seminoma and embryonal carcinoma. Forty testicular germ cell neoplasms were examined, which included 19 seminomas, three embryonal carcinomas, three teratomas, one yolk sac tumor, and 14 mixed germ cell tumors. The 14 cases of mixed germ cell tumors contained components of seminoma (n=7), embryonal carcinoma (n=11), teratoma (n=10), yolk sac tumor (n=2), and choriocarcinoma (n=1). All cases of pure seminoma and the seminomatous components of mixed germ cell tumors exhibited positive immunoreactivity for D2-40. Focal positivity for D2-40 was also observed in 29% of the embryonal carcinoma samples. D2-40 immunoreactivity in seminomas was characterized by diffuse membrane staining, whereas for embryonal carcinomas, staining was focal and distributed along the apical surfaces of the neoplastic cells. Immunohistochemical staining for KIT was observed in 92% of the seminoma samples and in none of the embryonal carcinomas. Conversely, CD30 expression was identified in 93% of the embryonal carcinoma samples and in none of the seminomas. Other germ cell components showed no immunoreactivity for D2-40, KIT, or CD30. KIT and CD30 are effective immunohistochemical markers in separating seminoma from embryonal carcinoma. Although a highly sensitive marker for seminomas, D2-40 positivity was also observed in a subset of embryonal carcinomas, thus limiting the utility of this antibody for discriminating between these two malignancies.