Germ cell tumors show many similarities to normal embryogenesis. This is, for example, illustrated by the expression of the marker of pluripotency, OCT3/4, known to play a pivotal role in the early stages of normal development. Interestingly, it is found to be the most informative diagnostic marker for the early developmental stages of malignant germ cell tumors. Expression regulation of OCT3/4 has been extensively studied in murine and human cell lines, including embryonic stem cell lines and tumor derived cell lines. We investigated for the first time the methylation status of the upstream region of the OCT3/4 gene in normal and neoplastic testicular primary tissues using bisulfite genomic sequencing. The cell line JKT-1, supposedly seminoma-derived, was included in the survey. Normal testis parenchyma, peripheral blood lymphocytes, spermatocytic seminoma, yolk sac tumor and teratoma, and JKT-1 showed a consistent hypermethylation. In contrast, seminoma and embryonal carcinoma were hypomethylated, confirmed by analyses after tumor micro-dissection. Testicular lymphomas showed the most heterogeneous pattern, although specific regions were consistently hypermethylated. In conclusion, the results obtained from this set of adult normal and neoplastic in vivo derived samples is in accordance to the in vitro data that expression of OCT3/4 is associated with specific changes in methylation. Moreover, the findings argue against OCT3/4 being a driving oncogenic factor in the pathogenesis of human germ cell tumors.