To study the mechanism of spermatogenesis during the premeiotic phase, a hybridoma producing monoclonal antibody (mAb) specific for early stages of spermatogenic cells was obtained. In immunohistochemical staining of adult testis, this mAb, designated as EE2, was able to react with type A to B spermatogonia and early meiotic cells, but not with Sertoli cells, Leydig cells, and other somatic tissues. Precursor cells of type A spermatogonia (gonocytes) were also positive for EE2 in perinatal mouse testis. The antigenic molecule recognized by mAb EE2 was a novel glycoprotein with molecular weight of 114 kDa, which had affinity with Con A and WGA lectins, and was susceptible to N‐glycanase, suggesting the presence of asparagine‐linked sugar chains. Furthermore, EE2 antigen was found to localize on the germ cell surface. The specific expression of this antigenic molecule suggests that it may play an important role in early spermatogenesis, of which only a little information is available at present. © 1995 Wiley‐Liss, Inc.