Chromosome band 9p21-22 is one of the most common targets for deletions in cancer. The p16 tumor suppressor gene maps to this region and is inactivated in a wide variety of tumor cell lines and primary tumors. However, in some of the neoplasms with frequent 9p21 loss of heterozygosity (LOH), a structurally and functionally normal p16 is found suggesting that this gene might not be the primary or only target for inactivation. To define the smallest region of overlap of deletions at chromosome band 9p21-22 and to clarify the involvement of p16, p15 and the IFN cluster gene in lymphoblastic leukemias, we used a multiplex polymerase chain reaction to construct a detailed map of deletions at 9p21-22. We studied DNA from 30 lymphoblastic leukemia patients and nine cell lines using 10 genes/markers that map to this region, including four STS markers located between p16 and D9S171 (STS1, STS2) or between p16 and IFNalpha (STS3, STS4). We found that the size of the deletions in this region is variable and that the commonly deleted region spans at least 400 kb; it includes the p16 gene but excludes the IFN gene cluster and the p15 gene. The identification of this commonly deleted region enables a more focused search for other putative tumor suppressor gene at 9p21 which could be relevant to leukemogenesis.