Several proteins, such as the placental/germ cell alkaline phosphatases (PLAPs), the stem cell factor receptor c-KIT, and the transcriptional regulator and marker of pluripotency OCT3/4, have been found in both normal immature and malignant germ cells, known as carcinoma in situ/intratubular germ cell neoplasia unclassified (CIS/ITGCNU). In the present study, immunohistochemical methods were used to evaluate the expression of these markers in a series of male gonads from fetuses from the second and third trimesters, and neonates. In addition to these markers, the presence of VASA (a protein specific for the germ cell lineage), TSPY (the testis-specific protein, Y-encoded), and the proliferation index (Ki-67 antigen) was analysed. All these proteins are reported to be present both during spermatogenesis and in CIS/ITGCNU. Positive staining for VASA with varying intensity was found in all germ cells, while TSPY was predominantly located in the prespermatogonial cells at all developmental ages. In contrast, the markers PLAP, c-KIT, OCT3/4, and Ki-67 were more frequent at earlier developmental stages and decreased gradually with time, although they could occasionally be detected in germ cells of neonates. These findings are in line with a declining number of gonocytes during fetal development, concomitant with an increase in the number of prespermatogonia. The latter have lost the immature germ cell phenotype. These findings are compatible with the hypothesis that CIS/ITGCNU arises from developmentally arrested germ cells, most likely primordial germ cells/gonocytes, at an early time point during intrauterine development. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.