The p16 gene, also referred to as MTS1, INK4, CDK4I, or CDKN2, at chromosome 9p21 has recently been described as a tumor suppressor that may be involved in a wide range of tumors. We have used a semiquantitative multiplex polymerase chain reaction assay to search for deletions of the p16 gene in 34 patients with chronic myeloid leukemia in blast crisis (CML BC), 19 patients with acute lymphoblastic leukemia (ALL), and 25 patients with acute myeloid leukemia (AML). Homozygous deletions of p16 exons were found in 5 of 10 (50%) patients with CML in lymphoid BC and in 5 (26%) ALL patients, but in only 1 (2%) case with AML. No deletions were found in CML BC of nonlymphoid phenotype. Comparison of chronic phase DNA or remission DNA with acute leukemia DNA in 5 individuals showed that the p16 deletions were acquired and not inherited, directly implicating these lesions in the pathogenesis of the disease. We conclude that functional elimination of the p16 gene, or a closely mapping gene, is involved in a significant number of patients with CML in lymphoid transformation.