Differences in the methylation patterns of male and female gamete DNA are likely to be involved in genomic imprinting. However, little is known of the mechanisms that regulate de novo methylation and demethylation during gametogenesis. We report here that the well-characterized M(r) 190,000 form of DNA methyltransferase (the only known form) is present in isolated mitotic, meiotic, and postmeiotic male germ cells, with the exception of meiotic pachytene spermatocytes, where the protein is undetectable by immunoblot analysis and a novel 6.2-kb DNA methyltransferase transcript is present. Whereas replication and methylation are coupled in somatic cells, the presence of DNA methyltransferase in postreplicative male germ cells is consistent with previously observed de novo methylation events in these cells. Immunofluorescence experiments revealed that DNA methyltransferase is localized to the nuclei of male germ cells, with a subset of spermatogonia and postreplicative leptotene/zygotene spermatocytes displaying prominent nuclear foci that are strongly enriched in DNA methyltransferase. The data suggest that down-regulation of DNA methyltransferase expression during the pachytene stage of meiosis utilizes an mechanism that is associated with the production of a larger mRNA, and that de novo methylation in leptotene/zygotene spermatocytes may take place in spatially restricted nuclear domains that are enriched in DNA methyltransferase.