Philadelphia chromosome-positive (Ph1) acute leukemia is a heterogeneous subset of acute leukemia with a poor prognosis. We studied five patients to determine the potential for phenotypic and molecular heterogeneity. Cellular characterization studies included light myeloperoxidase (L-MPO), terminal deoxynucleotidyl transferase (TdT), ultrastructural MPO (U-MPO), and immunophenotyping by flow cytometry using T11, T3, T4, T8, Leu 1, B1, Leu 12, HLA-DR (la), CALLA (J5), OKM1, My4, My7, My8, My9, and My10. DNA was analyzed for rearrangements of the breakpoint cluster region (bcr), immunoglobulin heavy chain, joining region (JH), immunoglobulin kappa light chain constant region (C kappa), and T cell receptor (TcR beta). RNA dot blots were hybridized by using molecular probes for MPO and TdT. We found that four of five cases were acute mixed-lineage leukemia (AMLL). One patient had acute unclassifiable leukemia. Of the four patients classified as having AMLL, three showed myeloid and lymphoid features, with one patient showing myeloid, T cell, and B cell features. The last case showed T cell and B cell features only. In one patient MPO/RNA was positive in spite of insufficient L-MPO or U-MPO to diagnose acute myelogenous leukemia (AML), thereby suggesting significant MPO gene expression before the production of sufficient MPO protein to meet the French-American-British criteria for AML. Three of the five patients showed rearrangement of bcr (cases 1, 2, and 5). Studies of these five patients support the concepts of molecular and phenotypic heterogeneity in Ph1 acute leukemia, demonstrate a high incidence of AMLL in this subset of acute leukemia, and support the use of lineage-associated molecular probes to define lineage at an earlier stage than previously possible.