Retinoic acid receptor B2 (RARB2) is frequently inactivated in cancer. Methylation in the S'-untranslated region and first exon is known to play a role; however, few studies have analyzed the detailed methylation pattern of the promoter region. We show that hypo- and hypermethylated alleles coexist in 5/11 cell lines in which RARB2 is inactivated. We present evidence supporting the mitotic transmission of these divergent methylation patterns and find a correlation between methylation divergence and heterozygosity at the 3p24 loci, suggesting an allelic methylation bias in these lines. Using a newly devised strategy based on allelic identification via methylation-sensitive restriction enzyme digestion combined with the use of a single nucleotide polymorphism, rs755661, we demonstrate that such a bias exists in three cancer cell specimens heterozygous at rs755661 and therefore amenable to this study. This previously unreported phenomenon of allelic methylation bias suggests that a promoter methylation-independent mechanism may be responsible for inactivation at the hypomethylated allele and this inactivation is reminiscent of an aberrant form of de novo imprinting. Approaches to interpreting methylation data should incorporate the notion of allelic methylation bias. (c) 2008 Wiley-Liss, Inc.