The reprogramming of DNA methylation in early embryos has been considered to be essential for the reprogramming to totipotency, the embryonic genome activation (EGA) and subsequent development. However, its degree appears to differ as a function of species and it may be altered by the in vitro enviromnent. While the rabbit is a pertinent mode!for species with a delayed EGA because both in vivo and in vitro developed embryos are easily available, the status of DNA methylation levels in both parental genomes after fertilization remains controversial. In order to generate precise data on the DNA methylation status in rabbit zygotes, we first of ali defined five pronuclear (PN) stages during the first cell cycle, which we subsequently used to classizy in vivo and in vitro developed rabbit zygotes. This allowed us to precisely quantizy the methylated DNA during the one cell stage, as weil as the total DNA content, whlch was used for normalization. We thus showed that both pronuclei display distinct patterns of DNA methylation reprogramming. In the maternai pronucleus (MP) the methylation leve!remains constant throughout the one cell stage, thanks to maintenance methylation during the S-phase. Conversely, in the paternal pronucleus (PP) partial demethylation occurs bcforc replication, probably as a result of active DNA demethylation, while maintenance methylation subsequent!y talœs place during the S-phase. Interestingly, we showed that PP DNA methylation reprogramming is partially altered by the in vitro enviromnent. Taken together, our approach evidences that rabbit is one of the species displaying partial DNA demethylation in the PP, and for the first time demonstrates maintenance methylation activity in both pronuclei during the first S phase.