We have studied growth, function, and phenotype of purified NK and T cells in long-term cultures and compared these parameters to those of conventionally prepared interleukin-2-activated lymphocytes with killer cell activity (LAK). Enrichment of NK and T cells was achieved by Percoll density gradient. Growth was analyzed by cell counts and [3H]TdR uptake. Cytotoxicity and tumor-binding efficacy were assessed in a 3-h 51Cr and single cell agarose assay, respectively, against K-562, Daudi, human ovarian cell line Ovcar-3, and fresh leukemic blasts. We found that long-term proliferation and lytic activity were highest in NK-enriched and lowest in T-enriched cultures. Conventional LAK cultures generated medium cytotoxicity levels. Lytic activity declined within 3 weeks in cultures not enriched for NK cells, while NK-enriched cultures showed high levels of cytotoxicity up to 6 weeks. No change was found in binding activity within 3 weeks with the exception of T cell-enriched fraction. A number of changes in the phenotypic patterns was observed in IL-2 cultures; the CD56+/CD3-/+ and CD56+/CD8+ subset increased in most cultures, whereas the CD56-/CD3+ subset decreased over time. The highly enriched NK cell culture maintained its NK cell phenotype over 5-6 weeks. We also delineated the most cytotoxic lymphocyte subset in long-term IL-2 cultures by complement dependent cytotoxic assays and fluorescence-activated cell sorting (FACS). Lytic activity in conventional LAK as well as in T and NK cell-enriched IL-2 cultures was mediated primarily by CD56+, CD16+, CD3- NK cells. The clinical implication of these studies is discussed.