This study investigates the prevalence and mechanism of non-CpG methylation in embryonic stem (ES) cells. Using a dual-label nearest neighbor analysis (NNA) and bisulphite genomic sequencing, the authors found that non-CpG methylation, primarily at CpA and to a lesser extent at CpT, is significantly higher in ES cells compared to somatic tissues. This non-CpG methylation is not caused by DNA methyltransferase 1 (Dnmt1) but is likely mediated by de novo methyltransferase Dnmt3a, which is highly expressed in ES cells. The study also demonstrates that Dnmt3a can induce methylation at CpA and CpT in Drosophila, supporting its role in non-CpG methylation. The functional significance of non-CpG methylation remains unclear, but the findings suggest that it may play a role in early development through reiterative de novo methylation at specific sites. The authors propose a model for the establishment of DNA methylation in mammalian development, where Dnmt3a is responsible for de novo methylation in early postimplantation stages, while Dnmt1 supports maintenance methylation in later stages.This study investigates the prevalence and mechanism of non-CpG methylation in embryonic stem (ES) cells. Using a dual-label nearest neighbor analysis (NNA) and bisulphite genomic sequencing, the authors found that non-CpG methylation, primarily at CpA and to a lesser extent at CpT, is significantly higher in ES cells compared to somatic tissues. This non-CpG methylation is not caused by DNA methyltransferase 1 (Dnmt1) but is likely mediated by de novo methyltransferase Dnmt3a, which is highly expressed in ES cells. The study also demonstrates that Dnmt3a can induce methylation at CpA and CpT in Drosophila, supporting its role in non-CpG methylation. The functional significance of non-CpG methylation remains unclear, but the findings suggest that it may play a role in early development through reiterative de novo methylation at specific sites. The authors propose a model for the establishment of DNA methylation in mammalian development, where Dnmt3a is responsible for de novo methylation in early postimplantation stages, while Dnmt1 supports maintenance methylation in later stages.