We determined concentrations of alpha and gamma-enolases in normal testis and in seminoma tissues by enzyme immunoassay. Concentrations of alpha-enolase were 4,170 +/- 2,040 ng/mg protein in normal testis (n = 8) and 8,140 +/- 4,480 ng/mg protein in seminoma (n = 8). Concentrations of gamma-enolase in seminoma (460 +/- 571 ng/mg protein) were significantly higher than those of normal testis (59 +/- 15 ng/mg protein). Immunohistochemistry showed positive tumor cells for gamma-enolase in 6 of 8 seminoma cases (75%). Serum gamma-enolase levels were elevated (greater than 6.0 ng/ml) in 9 of 12 patients (75%) with seminoma: 60% of stage I, and 100% of stages II and III. In 10 patients treated by surgical excision and chemotherapy, serum gamma-enolase was significantly reduced after the treatment. These findings indicate that elevated serum gamma-enolase is derived from enhanced gamma-enolase in seminoma tissues, and that serum gamma-enolase could be a useful biomarker for staging and monitoring clinical course in patients with seminoma.