In this report, we show that in vitro stimulation of human peripheral blood mononuclear cells (PBMC) with B lymphoblastoid cell lines results in preferential proliferation of cells with the phenotypic, genotypic and functional characteristics of natural killer (NK) cells. This culture system offers a useful method for obtaining large numbers of pure NK cells on which biochemical, molecular and functional studies can be performed. Using this culture system, an average 25-fold increase in NK cell number is obtained, whereas the number of T cells is increased only 3-fold. At early times, activation of both T and NK cells occurs, as detected by the presence of activation antigens on both cell types, but actual proliferation of NK cells starts on day 6 of culture. Elimination of CD3(+)/CD5(+) T cells from the cultured cells gives homogeneous preparations of large numbers of CD16(+)/NKH-1(+) cells that have the morphology of large granular lymphocytes, are powerful effectors of both spontaneous and antibody-dependent cell-mediated cytotoxicity, and rapidly proliferate in the presence of recombinant interleukin 2 (rIL-2). As fresh NK cells, NK cell-enriched preparations from 10-day cultures of Daudi-stimulated PBMC do not show rearrangement of the gene for the .beta.-chain of the T cell antigen receptor; the 1.3-kb functional transcript of the .beta.-chain gene was not expressed in NK cells, but the 1.0-kb truncated transcript was present in all preparations. Our data indicate that proliferation of both T and NK cells is dependent upon IL-2 production in the culture, because an anti-IL-2 antiserum completely suppresses proliferation. Because T cells, and in particular CD4(+) T cells, are required for preferential proliferation of NK cells, the NK cell stimulation induced by the B cell line is probably in part indirect, and due to induction of IL-2 production by allogeneic stimulation of CD4(+) cells. However, the B cell lines also need to interact directly with NK cells because neither allogeneic PBMC nor high doses of rIL-2 are sufficient to induce preferential proliferation of NK cells.