Dysregulation of cell cycle is important in oncogenesis. We analyzed the inactivation of the INK4 family CKI/CDK/RB pathway by gene promoter hypermethylation in leukemogenesis. The methylation-specific polymerase chain reaction (MSP) with primers for methylated (M-MSP) and unmethylated (U-MSP) alleles of the p15, p16, p18, and RB genes was used to study five leukemic cell lines, 50 acute myeloid leukemia (AML) and 25 acute lymphoblastic leukemia (ALL) samples. None of the leukemic cell lines showed p18 and RB methylation. p15 was methylated in Raji, while p16 was methylated in U937 and Raji. In NB4 and Jurkat, both alleles of p15 and p16 appeared to be deleted. At diagnosis, p15 methylation occurred in 29 (58%) AML patients, and 10 (40.0%) ALL patients. p16 methylation occurred in two (4%) AML and two (8%) ALL patients. Only one each of AML and ALL patients had concurrent p15 and p16 methylation. None of the patients had methylation of p18 or RB. In AML, p15 methylation was associated with M2 subtype ( p=0.018). Patients with and without p15 methylation had similar complete remission (CR) rates and projected 5-year overall survival (OS) or disease-free survival (DFS). Therefore, methylation inactivation of the INK4/CDK/RB pathway in leukemia involved primarily p15 and occasionally p16, but not p18 or RB. In AML, p15 gene methylation was associated with the M2 subtype, but was not prognostic for CR, OS, or DFS.