The formation of CD8+ killer cells from nonlytic thymocyte precursors is mediated by interleukin 2 and a cytokine termed CTL differentiation factor (CDF). While several reports have focused on the effects of recombinant molecules on the development of CTL, the natural protein responsible for CTL development that is produced by normal leukocytes has not been conclusively identified. A 24 kD native protein with CDF activity was enriched from leukocyte conditioned medium and neutralizing antibodies were produced. Utilizing immunoaffinity chromatography and reverse phase chromatography, we purified this CDF to homogeneity. All 21 amino acid residues at the NH2-terminus of CDF were found to be identical to that of IL-6. Natural CDF and IL-6 share many of the same biological properties, including costimulation of thymocyte proliferation with IL-1. Antibodies against CDF or IL-6 can block the activity of either cytokine, and anti-CDF blocks the activity of bulk leukocyte conditioned medium. These results indicate that IL-6 is the principal CTL differentiation factor produced by stimulated human leukocytes.