Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) patients consistently show a rearrangement in a 5.8-kilobase length of chromosome 22, referred to as the breakpoint cluster region (bcr). In Ph1-positive acute lymphoblastic leukemia (ALL), the breakpoint in chromosome 22 is more heterogeneous and, in some instances, does not occur within this region. In such cases the cell of origin of the neoplastic clone and the relationship of the disease to CML has remained obscure. We have analyzed the bcr rearrangement in the malignant cells from three patients who presented with Ph1-positive ALL and who in cytogenetic studies had shown evidence of variable involvement of myeloid cells in the Ph1-positive clone. Rearrangements in bcr typical of most cases of CML were detected in purified granulocyte preparations from two of the ALL patients (nos. 1 and 2) and in the blasts from patient 3 at the time of her terminal relapse. In the same analysis the simultaneously obtained granulocytes from patient 3, however, did not show any evidence of bcr rearrangement. Patient 3 was also heterozygous for the BamHI polymorphism in the X- linked hypoxanthine phosphoribosyltransferase (HPRT) gene, thus permitting a different method of clonal analysis based on methylation differences in active and inactive alleles. When DNA from her granulocytes that had shown no bcr rearrangement was hybridized to an HPRT probe, a pattern typical of a polyclonal population was seen. A similar pattern was exhibited by her marrow fibroblasts. In marked contrast, her simultaneously isolated blasts showed an unambiguous monoclonal pattern. These findings demonstrate the origin of the disease in the first two patients in a cell with myelopoietic as well as lymphopoietic potential and confirm the restricted lymphoid cell origin of the neoplastic clone in the third Ph1-positive ALL patient. Furthermore, they indicate that different target cells for transformation within the hematopoietic system may be affected by very similar bcr rearrangements.