The wild-type (wt) p53 tumor suppressor gene is commonly inactivated in human malignancies, either by mutations or by loss of expression. An additional proposed mechanism for inactivation of wt-p53 is amplification of the murine double minute 2 (MDM2) gene and overexpression of the MDM2 protein, which binds to p53 and eliminates its tumor suppressor function. To investigate a potential role for MDM2 in the inactivation of wt-p53 in pediatric acute lymphoblastic leukemia (ALL), we examined the expression of MDM2 and p53, as well as the occurrence of p53 mutations and possible amplification of the MDM2 gene, in 19 pediatric ALL cell lines and one pediatric acute myelogenous leukemia (AML) line. Although we did not find significant amplification of the MDM2 gene in any of the leukemic lines, we detected overexpression of MDM2 in all 10 lines that expressed wt-p53. Of the 10 lines without overexpression of the MDM2 gene, six (including the AML line) did not express p53, and four expressed mutant p53 with single point mutations in exons 7 and 8. To determine whether primary leukemic cells showed a similar correlation, we analyzed the original cryopreserved leukemic bone marrow cells from seven patients from whom cell lines were established. We obtained similar results from both the primary leukemic cells and the corresponding cell lines: overexpression of MDM2 was present in primary cells that expressed wt-p53 but not in cells that lacked expression of wt-p53. These findings suggest an important role for MDM2 in the pathogenesis of pediatric ALL in which leukemic cells express wt-p53.