Bone marrow samples from normal adults and children with nonhematologic malignancies not involving the marrow, acute lymphoblastic leukemia (ALL) in continued remission, and immune cytopenias were studied by two- color immunofluorescence (IF) and flow cytometry to characterize common acute lymphoblastic leukemia antigen (CALLA)-positive marrow lymphoid cells. Marrow was separated by Ficoll/Hypaque centrifugation followed by passage over a monoclonal antibody affinity column to remove myeloid cells prior to IF staining. A higher proportion of CALLA-positive cells was found in the pediatric marrows (mean, 33.7% +/- 6.3% SEM) than in the adult marrows (mean, 4.5% +/- 1.6% SEM). Two subpopulations of CALLA-positive cells identified by cell sorting to be of lymphoid morphology were found in both adult and pediatric marrows. A small subpopulation comprising 12.3% of the total CALLA-positive cells was characterized by a high intensity of CALLA, terminal deoxynucleotidyl transferase (TdT), and MY10 expression, but low B1, common leukocyte antigen, and peanut agglutinin receptor expression. The remainder of the CALLA-positive cells displayed low intensity of CALLA expression, positivity for the common leukocyte antigen, B1 and peanut agglutinin, and negative reaction with TdT and MY10. Both CALLA-positive subpopulations were positive for HLA-DR and the pan-B cell marker B4, but negative for cytoplasmic immunoglobulin, B2 and Leu 1. BrdU labelling studies showed that a similar proportion of cells in each subpopulation was in S phase. A slightly higher proportion of the strongly CALLA-positive cells possessed the morphologic features of high nuclear/cytoplasmic (N/C) ratio and prominent nucleoli. These studies suggest that a discrete maturation step occurs among CALLA- positive marrow lymphoid cells, resulting in the loss of TdT and MY10 expression, but gradual acquisition of the B cell marker B1 and the common leukocyte antigen. The presence of B4 antigen in nearly all CALLA-positive cells suggests that both subpopulations of normal CALLA- positive marrow cells are committed to the B cell lineage.