Abstract: Hypomethylation of DNA repeats, including satellite 2 DNA (Sat2), is one of the most frequent epigenetic changes in cancer. We examined ovarian epithelial tumors and diverse control tissues for methylation on only one strand (hemimethylation), both strands (symmetrical methylation), or neither strand at Sat2 CpG dyads using hairpin genomic sequencing. Analysis of the resulting cloned DNA molecules indicated that although carcinomas displayed much symmetrical hypomethylation of CpG dyads, there was cancer-linked hypermethylation at one of the thirteen dyads in the examined 0.2-kb Sat2 region. Hemimethylated sites were seen in both carcinomas and controls but, importantly, in carcinoma DNA molecules, they were significantly more likely to occur in clusters displaying the same orientation (the same strand methylated). Our data suggest that hemimethylated CpG dyads are intermediates in active demethylation during carcinogenesis and not just due to a failure of maintenance methylation during replicative DNA synthesis. Constitutive heterochromatin may be especially suitable for providing a snapshot of demethylation intermediates because hemimethylation might be more long-lived in heterochromatin due to its highly condensed state.