Components of calf thymus extract dialysable under acid conditions contained two natural killer (NK) cytotoxicity-stimulating factors, CSFa and CSFb, which could be separated by ion exchange chromatography. NK cytotoxicity of human peripheral blood mononuclear cells (PBMC) against human K562 tumour cells was strongly enhanced after 72 h pre-incubation with the factors. The CSFa/b-specific stimulation of PBMC required the presence of monocytes. The cytotoxic effector cells activated during pre-incubation of PBMC with the CSF were identified as monocytes and as NK cells present in the fraction of large granular lymphocytes (LGL). Selective cell depletion studies with LGL-containing subpopulations (free of monocytes) allowed factor-specific discrimination of the activated LGL. Pre-incubation of PBMC with CSFa stimulated NK cytotoxicity of LGL (Leu 7+11-; T8-), whereas pre-incubation with CSFb resulted in stimulation of LGL (Leu 7+11-; T8+). The biological effects of CSFa and CSFb could be further distinguished by analysis of surface marker expression during incubation of PBMC. CSFb scarcely influenced T4 expression, but strongly enhanced the expression of T8 and that of transferrin receptor, whereas CSFa had no significant influence on the expression of these three surface markers. Both factors induced a drastic reduction of tumour take incidence or tumour development in mice when applied before and after tumour challenge.