Clones capable of lysing fresh, uncultured tumor cells ("lymphokine-activated killer": "LAK" activity) were selected from microcultures derived from either E-rosette-positive or E-rosette-negative cell populations. All the selected clones displayed a strong cytolytic activity against the NK-sensitive K562 cell line. Two major phenotypic groups of clones could be identified: a first group expressed the CD3 differentiation antigen, present exclusively on mature T lymphocytes; however, in contrast to typical cytolytic T lymphocytes, the majority of these clones expressed the unusual CD4- CD8- phenotype, whereas the remainder were CD4- CD8+. A second group was represented by CD3- clones which, in most instances, expressed the T-cell-lineage-specific CD2 antigen. Following stimulation with phytohemagglutinin (PHA), most of the CD3+ LAK clones produced Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) whereas those expressing the CD3- phenotype did not. Since previous studies indicated that PHA may be inefficient in inducing lymphokine production by T-cell variants lacking the CD3/T cell receptor complex (TCR), CD3- clones were further stimulated with the calcium ionophore A23187 plus phorbol 12-myristate 13-acetate (PMA). Only 2/11 CD3- LAK clones produced small amounts of IL-2, whereas the majority released IFN-gamma. Given the peculiar phenotypic and functional properties of many CD3 + LAK clones, we suggest that they may belong to a T-cell subset distinct from typical CTLs.