Here, the role of methylation in regulation of rat Oct4 gene was evaluated during embryonic development, in adult tissues and in embryo-derived cells. First, the region 3.1 kb upstream to the rat Oct4 ATG site was cloned and sequenced. The rat Oct4 upstream sequence was similar to that in bovine, mouse and human with two upstream elements: proximal (PE) and distal enhancers (DE) and four homology conserved regions (CR1-4). The conserved regions in the rat have 69% - 96% homology with bovine, human, mouse sequences. Next, the methylation pattern in the promoter was determined during embryonic development, in adult tissues, in rat embryonic stem cell (ESC)-like cells and umbilical cord-derived cells (the feeder for ESC-like cells) using the bisulfite method and DNA sequencing. The promoter was methylated in adult and fetal tissues, and in days post coitus (DPC) 10.5 and 12.5 embryos and hypomethylated in DPC4.5 embryos and in rat ESC-like cells. The expression of Oct4 was evaluated by qRT-PCR. DPC4.5 embryos and rat ESC-like cells had higher expression of the Oct4 gene compared to DPC10.5 and 12.5 embryos, adult tissues and embryoid bodies derived from rat ESC-like cells. Thus, the methylation status correlated with the qRT-PCR results. These results indicate that the rat Oct4 3.1kb promoter region is organized and contains transcription binding and regulatory sites similar to those described for bovine, mouse and human. The rat Oct4 promoter is methylated during embryonic development after 4.5 DPC and during differentiation of rat ESC-like cells to embryoid bodies.