Although global erasure of DNA methylation has been observed in zygotes and primordial germ cells, the responsible enzyme(s) have been elusive. The demonstration that members of the Tet (ten eleven translocation) family of proteins are capable of catalyzing conversion of 5-methylcytosine (5mC) of DNA to 5-hydroxymethylcytosine (5hmC) raises the possibility that Tet proteins may participate in this process. Indeed, recent studies have implicated the involvement of Tet3 in the conversion of 5mC to 5hmC in zygotes. This result, combined with the demonstration that Tet proteins can further oxidize 5hmC to 5-carboxylcytosine followed by excision by thymine-DNA glycosylase, raises the possibility that active demethylation may take place in a process that involves Tet3-mediated oxidation followed by base excision repair. We demonstrated by immunostaining of mitotic chromosome spreads of preimplantation embryos that the 5hmC associated with the paternal genome in zygotes is gradually lost during preimplantation development. Our study suggests that, although the conversion of 5mC to 5hmC in zygotes is an enzyme-catalyzed process, loss of 5hmC during preimplantation appears to be a DNA replication-dependent passive process.