Genomic levels of DNA methylation undergo widespread alterations in early embryonic development. However, changes in embryonic methylation have proven difficult to study at the level of single-copy genes due to the small amount of tissue available for assay. This study provides the first detailed analysis of the methylation state of a tissue-specific gene through early development and differentiation. Using bisulfite sequencing, we mapped the methylation profile of the tissue-specific mouse skeletal alpha-actin promoter at all stages of development, from gametes to postimplantation embryos. We show that the alpha-actin promoter, which is fully methylated in the sperm and essentially unmethylated in the oocyte, undergoes a general demethylation from morula to blastocyst stages, although the blastula is not completely demethylated. Remethylation of the alpha-actin promoter occurs after implantation in a stochastic pattern, with some molecules being extensively methylated and others sparsely methylated. Moreover, we demonstrate that tissue-specific expression of the skeletal alpha-actin gene in the adult mouse does not correlate with the methylation state of the promoter, as we find a similar low level of methylation in both expressing and one of the two nonexpressing tissues tested. However, a subset of CpG sites within the skeletal alpha-actin promoter are preferentially methylated in liver, a nonexpressing tissue.