An immunohistochemical study was performed in 120 cases of mediastinal and testicular germ cell tumors from archival, paraffin-embedded material to compare the patterns of expression between the two groups with a panel of markers, including broad-spectrum keratin, CAM 5.2 low-molecular-weight cytokeratin, placental-like alkaline phosphatase (PLAP), alpha-fetoprotein (AFP), human beta-chorionic gonadotropin (hCG), vimentin, and CD30 (Ki-1 antigen). Significant differences were observed between mediastinal and testicular seminomas: mediastinal seminomas showed strong dot-like paranuclear positivity of the tumor cells with antibodies to CAM 5.2 low-molecular-weight keratin in 80% of cases (32 of 40), as compared with only 20% positivity (5 of 24) in testicular seminomas; placental alkaline phosphatase (PLAP) was also found to be less commonly expressed in testicular seminomas (12 of 24) than in mediastinal seminomas (37 of 40); a similar pattern of expression was also observed for vimentin, which was present in scattered tumor cells in a higher proportion of mediastinal seminomas (28 of 40) than in testicular seminomas (11 of 24). The staining pattern and distribution of these markers did not show significant differences between the two groups for the various other tumor categories studied, including yolk sac tumor, embryonal carcinoma, and choriocarcinoma. The tumor cells in both testicular and mediastinal embryonal carcinoma showed strong positivity for the CD30 antigen; however, strong positivity for this marker was also observed in 6 of 25 yolk sac tumors and in scattered individual tumor cells in 4 of 63 seminomas. The results of this study show that significant differences exist between the immunostaining patterns of mediastinal and testicular seminomas, suggesting that the former may be characterized by a more mature phenotype than their testicular counterparts. Also, CD30 expression may not be necessarily restricted to embryonal carcinomas and occasionally may be observed in yolk sac tumors and seminoma cells, supporting the close histogenetic relationship that exists among these tumor types.