The cyclin-dependent kinase 4-inhibitor (CDK41; p16; or MTS1) gene has been proposed as a candidate for a tumor-suppressor gene located in chromosome 9p21, a frequently deleted region in a wide spectrum of human cancers, including leukemias. Recent studies disclosed that it was frequently deleted or mutated in a variety of primary human cancers, including acute lymphoblastic leukemia. The purpose of this study is to figure out the precise manners and frequencies of p16 gene inactivation in diverse hematopoietic tumor types and thus to clarify its significance in development of human hematopoietic malignancies. A total of 410 tumor specimens from patients with primary hematopoietic malignancies were examined for deletions of the p16 gene as well as the neighboring p15 gene and the nearby interferon alpha gene by Southern blot analysis. Tumor-specific mutations or small deletions of the p16 gene were also studied in 74 patients using single-strand conformation polymorphism analysis and direct sequencing. Loss of the p16 gene was most frequently observed among the three genes examined and was found in 59 of the 410 patients: 2 of 134 with acute myelocytic leukemia, 41 of 105 with acute lymphocytic leukemia, 2 of 15 with chronic lymphocytic leukemia, 5 of 14 with adult T-cell leukemia, 4 of 33 with non-Hodgkin's lymphoma, 3 of 8 with mixed-lineage leukemia, and 2 of 61 with chronic myelocytic leukemia. In 16 of the 59 patients, the p16 deletions occurred due to rearrangements within the small region between the p15 exon 2 and the p16 exon 2. Tumor-specific mutations or small deletions of the p16 gene were not detected in the 74 patients examined, including 12 of 14 patients with hemizygous deletions of the gene. Loss of the p16 gene is frequent in and highly specific to lymphoid malignancies (54 of 183 [30%] in lymphoid tumor v2 of 219 [1%] in myeloid tumors; P < .0001). The deletion analyses strongly suggest that the p16 gene is a tumor-suppressor gene located in chromosome 9p21 that is involved in development of human lymphoid tumors. Gene deletions but not minute mutations should be the predominant mechanism of p16 gene inactivation in these types of tumors.