The p16INK4A tumor suppressor gene is frequently disrupted by mutation or deletion in a wide range of cancer types, ranging from leukemia to cancers of the bladder, skin, lung, liver, and spleen. We have previously shown that deletion of at least one copy of the p16INK4A gene is associated with an increased risk of relapse in pediatric leukemia. Our data suggest that hemizygous p16INK4A deletion may be constitutional, conferring susceptibility to leukemia. Confirmation of this association is worthy of a larger study. Data from primary leukemia specimens are also presented here which examined the possibility that the remaining allele of the gene was inactivated by another mechanism such as mutation or was silenced by methylation. These possibilities were formally excluded in a case of hemizygous loss of the p16INK4A gene in leukemia, establishing that in this case the p16INK4A deletion was either semidominant or fully haploinsufficient for relapse susceptibility in this disease. Implementation of high throughput methods such as those used here for detecting hemizygous loss of tumor suppressor genes will become increasingly important for molecular diagnosis of cancer. This is particularly true for the emerging class of tumor suppressor genes where deletion of one allele is sufficient to confer cancer susceptibility or poor prognosis with standard treatment.