To define the clinical and biologic significance of childhood acute mixed-lineage leukemia diagnosed by stringent criteria, we studied 25 cases of acute lymphoblastic leukemia expressing greater than or equal to 2 myeloid-associated antigens (My+ ALL), and 16 cases of acute myeloid leukemia expressing greater than or equal to 2 lymphoid associated antigens (Ly+ AML). These cases represented 6.1% of 410 newly diagnosed ALLs (two treatment protocols) and 16.8% of 95 AMLs (two protocols). T-lineage--associated antigens were identified in 9 of the My+ ALL cases and in 14 of those classified as Ly+ AML; all but 1 of the 19 cases that could be subclassified had an early thymocyte stage of differentiation. The My+ ALL cases had an increased frequency of French-American-British (FAB) L2 morphology (36%); the Ly+ AML cases were characterized by FAB M1 or M2 morphology, low levels of myeloperoxidase reactivity and combined populations of myeloperoxidase-positive large blasts and small blasts generally of hand-mirror morphology. Karyotypic abnormalities included t(9;22)(q34;q11) in three cases of My+ ALL, 11q23 translocations in two cases of My+ ALL, and 14q32 translocations in three My+ ALL and five Ly+ AML cases. Mixed-lineage expression lacked prognostic significance in either ALL or AML; however, the findings indicate that some patients with Ly+ AML may respond to prednisone, vincristine, and L-asparaginase after failing on protocols for myeloid leukemia. At relapse, two My+ ALLs had converted to AML and two Ly+ AMLs to ALL; one case in each group showed complete replacement of the original karyotype. Acute mixed-lineage leukemia does not adequately describe the heterogeneity of the cases identified in this study and should be replaced by a set of more restrictive terms that indicate the unique biologic features of these leukemias.