Aberrant methylation of multiple CpG islands has been described in acute myeloid leukemia (AML), but it is not known whether these are independent events or whether they reflect specific methylation defects in a subset of cases. To study this issue, the methylation status of 14 promoter-associated CpG islands was analyzed in 36 cases of AML previously characterized for estrogen-receptor methylation (ERM). Cases with methylation density of 10% or greater were considered positive. Seventeen cases (47%) were ERM(+) while 19 cases were ERM(-). Hypermethylation of any of the following, p15, p16, CACNA1G, MINT1, MINT2, MDR1, THBS1, and PTC1 (2 promoters), was relatively infrequent (6% to 31% of patients). For each of these CpG islands, the methylation density was positively correlated with ERM density (rank order correlation coefficients, 0.32-0.59; 2-tailed P < or = .058 for each gene). Hypermethylation of MYOD1, PITX2, GPR37, and SDC4 was frequently found in AML (47% to 64% of patients). For each of these genes as well, methylation density was positively correlated with ERM density (correlation coefficients 0.43 to 0.69, P < or = .0087 for each gene). MLH1 was unmethylated in all cases. Hypermethylation of p15, MDR1, and SDC4 correlated with reduced levels of expression. There was an inverse correlation between age and the number of genes methylated (P = .0030). It was concluded that CpG-island methylation in AML results from methylation defects in subsets of cases. These results have potential implications for the classification and prognosis of AML and for the identification of patients who may benefit from treatment with methylation inhibitors.