The promoter region of the cyclin-dependent kinase inhibitorp15^INK4Bcontains a CpG island that is hypermethylated in many hematologic malignancies. To explore the relationship between patterns of methylation and gene transcription, we used bisulfite genomic sequencing to obtain a detailed analysis of methylation in acute leukemia, leukemia cell lines, and normal lymphocytes. The entire CpG island region of p15 was largely devoid of methylation in normal lymphocytes, but methylation of varying density was found in primary acute leukemia. Methylation density was generally conserved between the alleles from each sample, but marked heterogeneity for the specific CpG sites methylated was observed. Patterns of methylation were compared and expression assessed with reverse-transcriptase polymerase chain reaction (RT-PCR). The density of methylation within the CpG island, and not any specific location, correlates best with transcriptional loss. Leukemias with methylation of approximately 40% of the CpG dinucleotides on each allele had complete gene silencing, with variable, but diminished expression with less dense CpG island methylation. Our results suggest that the transcriptional silencing of p15 in conjunction with aberrant hypermethylation is best understood as an evolutionary process that involves progressively increasing methylation of the entire p15CpG island.