The acquisition of genomic methylation in the male germ line is initiated prenatally in diploid gonocytes, while DNA methylation in the female germ line is initiated postnatally in growing oocytes. We compared the temporal expression patterns of the DNA methyltransferases, DNMT1, DNMT3a, DNMT3b, and DNMT3l in the male and female germ lines. DNMT1 expression was examined by immunocytochemistry and Northerns with an emphasis on the prenatal period. In the female, there is a gradual down-regulation of DNMT1 protein in prenatal meiotic prophase I oocytes that is not associated with the production of an untranslated transcript, as it is in the male; these results suggest that the mechanism of meiotic down-regulation differs between the sexes. In the male, DNMT1 is unlikely to play a role in the prenatal acquisition of germ line methylation patterns since it is down-regulated in gonocytes between 14.5 and 18.5 days of gestation and is absent at the time of initiation of DNA methylation. To search for candidate DNMTs that could be involved in establishing methylation patterns in both germ lines, real-time RT-PCR was used to simultaneously study the expression profiles of the three DNMT3 enzymes in developing testes and ovaries; DNMT1 expression was included as a control. Expression profiles of DNMT3a and DNMT3l provide support for an interaction of the two enzymes during prenatal germ cell development and de novo methylation in the male. DNMT3l is the predominant DNMT3 enzyme expressed at high levels in the postnatal female germ line at the time of acquisition of DNA methylation patterns. DNMT1 and DNMT3b expression levels peak concomitantly, shortly after birth in the male, consistent with a role in the maintenance of methylation patterns in proliferating spermatogonia. Together, the results provide clues to specific roles for the different DNMT family members in de novo and maintenance methylation in the developing testis and ovary.