The expression of POU5F1 is controlled by cis-regulatory elements located 5' upstream from the initiation start site. This regulatory region is highly conserved among species, and especially four conserved regions (CR1 to 4) have been identified. The minimal promoter is located in the CR1 region, while the proximal and distal enhancers involved in cell specific regulation of expression are located in CR2- 3 and CR4 regions respectively. In the mouse the two enhancer regions contribute differently to gene expression depending on the developmental stage of the embryo. POU5F1 repression of expression is induced by regulatory factors binding to the 5' upstream region but also by DNA and histone methylation. DNA methylation of POU5F1 has been mostly analyzed in in vitro derived stem cells. However, promotor DNA methylation levels have been shown to differ between embryonic stem cells and the "in embryo" counterparts they are derived from. We thus decided to analyze DNA methylation of the four conserved regions of the POU5F1 upstream region in the first embryonic lineages. Therefore we took benefit from the rabbit embryo whose epiblast, hypoblast and trophoblast can be easily isolated. At that stage, POU5F1 expression is restricted to the epiblast. We evidenced an hypomethylation of the four conserved regions of POU5F1 5' upstream region in the epiblast. Interestingly even the CR4 region which is supposed to be functional in the ICM but not in the epiblast conserved a very low methylation level in this tissue. CR1 methylation was lower in day 6 embryos than in fibroblasts.