Hypermethylation of tumor-suppressor genes has been implicated in the pathogenesis of human cancers. This study was designed to examine the methylation profiles of a selected group of p53 target genes (APAF-1, CASP-8, DAPK-1, IGFBP-3) and to correlate the findings with the histopathological characterization of testicular germ cell tumors (TGCT). Promoter methylation status was analysed by highly sensitive real-time methylation-specific PCR in 46 primary TGCTs (26 seminomas and 20 nonseminomas) and 15 normal testicular tissue samples. APAF-1 methylation was detected in all of the seminomatous and nonseminomatous TGCTs as well as in 60% of normal testicular tissue. Methylation of DAPK-1 was frequent in seminomas (50%) and nonseminomas (20%), but not in normal testicular tissue (6%). The degree of DAPK-1 methylation correlated with the clinical stage of the disease (P=0.05) and was useful in differentiating seminomatous from nonseminomatous, and malignant from nonmalignant testicular tissue (P=0.04 and 0.02, respectively). The APAF-1 methylation index achieved a highly significant differentiation between seminomatous or nonseminomatous tissue and nonmalignant testicular tissue (P=0.0001). In testicular tumorigenesis, promoter methylation of specific p53 target genes occurs at early stage but to varying degrees. Methylation also occurs in normal testicular tissue, which is in contrast to findings in other urogenital malignancies. Further studies will be necessary to determine whether the methylation level may be used as marker for risk estimation, especially in clinical stage I disease.