Imprinted genes are epigenetically marked during gametogenesis so that they are exclusively expressed from either the paternal or the maternal allele in offspring1. Imprinting prevents parthenogenesis in mammals and is often disrupted in congenital malformation syndromes, tumours and cloned animals1. Althoughde novoDNA methyltransferases of the Dnmt3 family are implicated in maternal imprinting2, the lethality ofDnmt3aandDnmt3bknockout mice3has precluded further studies. We here report the disruption ofDnmt3aandDnmt3bin germ cells, with their preservation in somatic cells, by conditional knockout technology4. Offspring fromDnmt3aconditional mutant females diein uteroand lack methylation and allele-specific expression at all maternally imprinted loci examined.Dnmt3aconditional mutant males show impaired spermatogenesis and lack methylation at two of three paternally imprinted loci examined in spermatogonia. By contrast,Dnmt3bconditional mutants and their offspring show no apparent phenotype. The phenotype ofDnmt3aconditional mutants is indistinguishable from that ofDnmt3Lknockout mice2,5, except for the discrepancy in methylation at one locus. These results indicate that both Dnmt3a and Dnmt3L are required for methylation of most imprinted loci in germ cells, but also suggest the involvement of other factors.