Although 14.5-day murine fetal liver (FL) has few, if any, mature natural killer (NK) cells, culture of FL with recombinant human IL-2 (rhIL-2) and stroma from irradiated NK longterm bone marrow cultures (NK-LTBMC) allows proliferation and differentiation of NK cell progenitors. Using this system, NK cell progenitors were found in both CD34+ and CD34- sorted subpopulations of FL. The CD34 antigen was expressed by 14+/-1.3% of whole FL cells, while mature NK cells cultured from NK cell precursors in FL did not express the CD34 antigen. Anti-TER-119 mAb reacted with 84%+/-10.3% of the FL cells, and NK cell progenitors were enriched in the TER-119- subpopulation. After coculture with rhIL-2 and stroma, neither TER-119- nor TER-119+ cells expressed antigens associated with T cells (CD3, CD4, and CD8) or myeloid cells (Gr-1 and Mac-1). Only the TER-119 subpopulation generated NK1.1+ (77%) and B220+ (87%) cells. Within the TER-119 subpopulation, both CD34+ and CD34- cells generated cytolytic and NK1.1+ cells after culture. By a limiting dilution assay (LDA) of the Lin (i.e., negative for NK1.1, CD3, CD4, CD8, B220, Gr-1, and TER-119) CD34 positive or negative subpopulations, the calculated mean frequency of NK cell progenitors was about 1/100 for the CD34+Lin- subpopulation and about 1/(200-300) for the CD34-Lin- subpopulation. In kinetic studies, we found that NK1.1 antigen expression continued to increase with time in culture for both the CD34+Lin- and CD34-Lin- fractions. In contrast, the percentage of CD34+ cells decreased rapidly and produced CD34- cells, and the CD34- population remained CD34-. These data suggest that both CD34+ and CD34- subpopulations of FL can differentiate into NK cells when cocultured for 13 days with irradiated NK-LTBMC stroma and rhIL-2, and that CD34+ progenitors differentiate to CD34- precursors, which in turn differentiate to CD34- mature NK cells.