M2A antigen is an oncofetal antigen associated with germ cell neoplasia, present in testis on fetal gonocytes and re-expressed on carcinoma in situ (CIS) and germ cell tumours. We developed a panel of monoclonal antibodies (mAb), M2A (IgG2a), D1-26 (IgG2b) and D2-40 (IgG1), to this antigen in order to characterize its structure and study its distribution among germ cell tumours. M2A antigen was purified by sequential lectin and antibody affinity chromatography and characterized as a monomeric M, 40 000 surface sialoglycoprotein, extensively glycosylated with O-linked carbohydrate structures, but devoid of N-linked sugars. Terminal sialic acid residues were required for reactivity with mAb M2A and D1-26, but not D2-40. Sections of 69 testicular germ cell tumours, fixed in formalin and embedded in paraffin, were stained with mAb D2-40 to examine the distribution of M2A antigen. Uniform membrane staining was observed in seminomas, and focal staining in 69% of embryonal carcinomas, 29% of teratomas and 25% of yolk sac tumours. CIS in the vicinity of all germ cell tumours also displayed uniform membrane staining. The characterization of M2A antigen, and the development of mAb which react with it in conventionally preserved archival specimens, provide important initiatives to study the origin and progression of germ cell neoplasia.