Multiple genes have been shown to be independently hypermethylated in lymphoid malignancies. We report here on the extent of concurrent methylation of E-cadherin, Dap-kinase, O(6)MGMT, p73, p16, p15 and p14 in 129 pediatric ALL cases. While most of these genes demonstrated methylation in a proportion of cases, O-6 MGMT, p16 and p14 were infrequently methylated (11, 7 and 3%, respectively). Methylation of at least one gene was found in the vast majority (83%) of cases. To determine the extent and concordance of methylation we calculated a methylation index (MI = number of methylated genes/number of studied genes) for each sample. The average MI was 0.28, corresponding to 2/7 methylated genes. MI was correlated with standard prognostic factors, including immunophenotype, age, sex, WBC and presence of specific translocations (TEL-AML1, BCR-ABL, E2A-PBX1 or MLL-AF4). We determined that children greater than or equal to10 years old and children presenting with high WBC (greater than or equal to50 x 10(9)/l) both associated with a higher MI (P<0.01 and <0.05, respectively). T-ALLs demonstrated a lower MI ( median = 0.17) than precursor B ALLs ( median = 0.28). Among the different molecular subgroups, MLL-ALLs had the highest MI ( mean = 0.35), while ALLs carrying the t( 1; 19) had the lowest MI ( mean = 0.07). The most common epigenetic lesion in childhood ALL was methylation of E-cadherin (72%) independent of the molecular subtype or other clinicopathological factors.