Silencing of the cyclin-dependent kinase inhibitor gene p15^INK4B by cytosine methylation of the promoter region has been associated with some types of hematological malignancy. To study in detail the patterns of p15^INK4B methylation in patients with acute myeloid leukemia, we adopted a novel approach based on PCR amplification of bisulfite-treated DNA followed by resolution of differentially methylated sequences by denaturing gradient gel electrophoresis. This method visually displays the degree and heterogeneity of DNA methylation and enables the isolation and characterization of distinct clonotypic epigenotypes. A surprisingly high degree of intra- and interindividual heterogeneity of p15^INK4B methylation was observed in the 65 acute myeloid leukemia patients examined. Methylation was detected in 46 (71%) of the patients and was observed more frequently in the French-American-British subtypes M1/M2 than in M4/M5 (P < 0.025). Examination of the same panel of samples using a highly sensitive methylation-specific PCR method showed methylated p15^INK4B alleles in 61 (94%) of the samples. We present evidence that the higher frequency of p15^INK4B methylation determined by methylation-specific PCR may, at least in part, be due to the presence of a small fraction of p15^INK4B-methylated lymphocytes in normal blood.