We report karyotypic analysis of 24 male germ cell tumors (GCTs) with clonally abnormal karyotypes biopsied from testicular and extragonadal lesions from 20 patients belonging t o the histologic categories seminoma, teratoma, embryonal carcinoma. choriocarcinoma, and endodermal sinus tumor. Chromosomes I, 7 , 9 , 12, I7,2 I, 22, and the X chromosome were nonrandomly gained in these tumors. Nonrandom structural changes affected most frequently chromosomes I and 12, the latter as i( I2p) and/or del( I2)(q I h q 2 2 ) . The i( I2p) was seen in 90% of tumors which included all histologic subtypes and gonadal as well as extragonadal presentation. Our present results, along with those from published data on fresh GCT biopsies, establish that i( I2p) is a highly nonrandom chromosome marker of all histologic as well as anatomic presentations of GCTs. In contrast,, we found del( I2)(q I h q 2 2 ) exclusively in nonseminomatous GCTs (NSGCTs) and mixed GCTs (MGCTs) occurring in 44% of such lesions. Because successful cytogenetic analysis of fresh tumor specimens is not always possible, we developed a method based on DNA analysis to detect i( I2p) as increased copy number of I2p. In addition t o the changes affecting chromosome I 2 identified above, we have detected, for the first time, cytological evidence of gene amplification in the form of homogeneously staining regions (HSRs) and double minute chromosomes (dmins) in treated as well as untreated primary extragonadal and metastatic GCTs and confirmed the presence of amplified DNA in one of these tumors at the molecular level by the in-gel renaturation method. Hybridization of DNA from cultured cells from an HSR-bearing tumor with a panel of probes for genes known t o be amplified or otherwise perturbed in diverse tumor systems did not identify the amplified gene, suggesting amplification of a novel gene or genes. This study comprises the largest series of GCT cytogenetics attempted so far. Notably, it includes data on a series of primary mediastinal tumors, a group which previously has not been studied in any detail.