Natural killer (NK) cells are bone marrow-derived large granular lymphocytes that express the CD56 surface antigen. The CD56bright NK subset represents approximately 10% of all NK cells and is thought to be the least differentiated NK cell component in blood. The most mature NK cell expresses CD56 at low density and CD16 (FcR gammaIII) at high density, whereas CD56bright NK cells either lack CD16 (CD56bright CD16-) or express it at low density (CD56bright CD16dim). c-kit is a tyrosine kinase receptor which is expressed on both CD34+ hematopoietic precursor cells and CD56bright NK cells. In the current study, we characterize interleukin (IL)-2 receptor (IL-2R) and c-kit expression in each of the CD56bright subsets. Both the CD56bright CD16- and CD56bright CD16dim NK subsets express the high-affinity IL-2R and the c-kit receptor when isolated from fresh blood. However, each CD56bright NK cell subset has distinct functional responses to IL-2, the c-kit ligand (KL), or both. Activation of the high-affinity IL-2R on CD56bright CD16- NK cells induces a proliferative response that is significantly weaker than that observed in the CD56bright CD16dim NK cell subset. Incubation of the CD56bright CD16 NK cell subset with KL significantly enhances IL-2-induced proliferation, while KL has no such effect on the CD56bright CD16dim NK subset. Activation of the high-affinity IL-2R in both CD56bright subsets induces lymphokine-activated killer (LAK) activity, but the addition of KL has no effect on LAK activity. Co-stimulation of either CD56bright subset with IL-12 and concentrations of IL-2 that only saturate the high-affinity IL-2R induces substantial interferon (IFN)-gamma production. The addition of KL to this co-stimulatory signal enhances IFN-gamma production in both CD56bright NK subsets. The distinct functional responses to IL-2 and KL seen in the CD56bright CD16- and CD56bright CD16dim NK subsets provide insight into IL-2R signaling and suggest that each phenotype identifies a discrete stage of NK cell differentiation.