In human spermatogenic cells, in contrast to somatic cells, expression of major histocompatibility complex (MHC) class I molecules is undetectable. This lack of expression may contribute to the absence of female immune reaction against spermatozoa and may be necessary for gamete fusion. Among the molecular repressor mechanisms that may be used at the DNA level, we investigated 5’ CpG methylation of the different class Ia and class Ib loci in meiotic pachytene spermatocytes and postmeiotic round spermatids, which had been purified from human testes by centrifugal elutriation. These results were compared with those obtained with mature spermatozoa and peripheral blood mononuclear cells. Using methylation-sensitive restriction enzymes and DNA locus-specific probes, we found that HLA-A, HLA-B/C, and HLA-E loci were similarly unmethylated in the germ and somatic cells tested, whereas HLA-F and HLA-G were even less methylated in the former cells. Together with the observation that spermatozoon DNA contains class I genes that are transfectable and able to direct transcription and protein synthesis in murine L cells, these data suggest that HLA class I genes are in an active conformation in male germ cells. We indeed found that both spermatocytes and spermatids contained low levels of class Ia and class Ib mRNA. Using reverse transcriptase-polymerase chain reaction, followed by DNA sequencing, we also detected three HLA-G transcriptional isoforms, resulting from alternative splicings, which suggested that this class Ib gene may have a potential function in these germ cells. Although intracellular expression of β2-microglobulin (the light chain that associates with HLA class I heavy chains) was found in spermatocytes but not in round spermatids, no membrane-bound nor intracellular translated HLA class I heavy chain was detected in either germ cell type, when monomorphic anti-HLA class I monoclonal antibodies were used. Thus, lack of expression of HLA class I proteins in the male germ line is likely to involve post-transcriptional mechanisms of regulation.