Intratubular malignant germ cells (ITMGC), as assessed by clinicopathologic or cytogenetic studies, are regarded as a preinvasive lesion of all human testicular germ cell tumors with the exception of yolk sac tumors (in infants) and spermatocytic seminomas. To characterize specific surface molecules of ITMGC, we raised three mouse monoclonal antibodies (mAb) against NCR-G3 (G3), a multipotent, human embryonal carcinoma (EC) cell line, and screened cryostat sections of human testicular tissue containing ITMGC. These three mAb (HB5, IgG1; HF2, IgG1; HE11, IgG1) reacted to the surface of ITMGC, seminomas, and EC in vivo as well as to human EC cell lines in vitro. Expression of HB5 and HF2 antigens was down-regulated during cellular differentiation of G3 cells by retinoic acid or N,N'-hexamethylene-bis-acetamide treatment, whereas that of HE11 antigen was up-regulated with cellular differentiation by retinoic acid. Furthermore, these three mAb reacted to stage-specific prespermatogonia in the human fetus but not in human adults. HB5, HF2, and HE11 antigens were shown to be glycoproteins with molecular weights of approximately 80, 80, and 70 kd, respectively, and could be immunoprecipitated after deglycosylation treatment. Peptide mapping with Staphylococcus aureus V8 protease suggested that the HB5 and HF2 antigens were identical. We concluded that HB5/HF2 and HE11 antigens are oncodevelopmental antigens in testicular germ cell tumors and human spermatogenesis that may play a significant role in tumorigenesis and the development of human germ cells.