Recently, it has been shown that the homozygous deletion of the cyclin- dependent kinase-4 inhibitor (CDK4I; p16) gene, which is mapped to chromosome 9p21, is frequently observed in a wide spectrum of human cancers, including leukemias. Therefore, the CDK4I gene is thought to be a putative tumor- suppressor gene. We report here that both alleles of the CDK4I gene were completely or partially deleted in human leukemia cells derived from both patients and established cell lines. Thirty-seven hematopoietic cell lines and samples from 72 patients with leukemias were examined for homozygous loss of the CDK4I gene locus by Southern blot analysis. We found that a part or the whole of the CDK4I gene was homozygously deleted in 14 of the 37 (38%) cell lines and 4 of 72 (6%) samples from leukemia patients, including 45 with acute myelocytic leukemia, 14 with acute lymphocytic leukemia (ALL), and 13 with chronic myelocytic leukemia in blastic crisis. In the cell lines, the homozygous deletion of the CDK4I gene was detected in a variety of cell lineages, whereas all 4 cases showing the homozygous deletion were confined to ALL. It should be noted that 2 of them had no cytogenetic abnormalities of chromosome 9. Our results suggest that loss of the CDK4I function may contribute to immortalization of human leukemia cells and play a causative role at least in development of human lymphocytic leukemias.