During detailed immunophenotypic analyses of marrow blasts from 336 acute lymphoblastic leukemia (ALL) patients, a very small percentage of cases reactive with B-cell-directed as well as T-cell-directed monoclonal antibodies (MoAbs) were identified. Five ALL cases were biphenotypic since they coexpressed CD2 (Tp50) and CD19 (Bp95) antigens at the single-cell level. The composite immunophenotype of these biphenotypic ALL cases was [TdT+HLA-ABC+CD2+CD3-CD10+CD13-CD14-CD16- CD19+CD20+ ++-CD21-CD33-CD34+Bgp95-C mu- slg-]. Low-molecular-weight B- cell growth factor (LMW-BCGF), recombinant interleukin-2 (rIL-2), and rIL-3 stimulated the proliferative activity of biphenotypic leukemic lymphocyte precursors without inducing differentiation. In the presence of the phorbol ester TPA, leukemic blasts from two cases differentiated along the B precursor pathway to the [CD2-CD10+CD19+CD20+C mu+slg-] pre- B cell stage. Biphenotypic ALL cases did not share a common configuration and gene rearrangement pattern of the immunoglobulin heavy chain genes or T-cell receptor (TCR) genes. Three cases had rearranged C mu genes but germline TCR genes, one case showed rearrangement of both C mu and TCR genes, and the remaining case had rearranged TCR genes but germline C mu genes. All five patients attained prompt remission after standard induction chemotherapy. Three to four years after initial diagnosis, four patients are now off chemotherapy and remain alive in their first remission. One patient relapsed at 3 years, 7 months, but promptly achieved complete remission after reinduction chemotherapy and remains in second remission off chemotherapy greater than 3 years after her reinduction therapy. With two-color immunofluorescence staining techniques and multiparameter flow cytometric analyses, we identified a small population of CD2+CD19+ lymphoid cells in fetal livers (FLs) and fetal bone marrows (FBMs), which may represent the putative normal counterparts of biphenotypic ALL blasts. A CD2+CD19+ normal biphenotypic lymphoid precursor cell line, designated FL 8.2 CD2+, was established from an FL of 8-weeks of gestational age by Epstein-Barr virus (EBV)-induced blastoid transformation. The composite immunophenotype of FL 8.2 CD2+ cell line was [TdT+HLA-ABC+HLA-DR+ CD2+CD5-CD7-CD10+/-CD13-CD19+CD20-CD21+ CD22+CD33-CD34+/-Bgp95-CDw40+C mu-slgD-slgM-]. FL 8.2 CD2+ cells showed germline patterns of immunoglobulin heavy-chain joining region, heavy- chain constant region, kappa light-chain constant region genes, and TCR beta-chain genes. Cross-linking of CD2 as well as CD19 antigens on FL 8.2 CD2+ cells caused an increase of intracellular ionized calcium.(ABSTRACT TRUNCATED AT 400 WORDS)