Cyclosporin A (CSA) is commonly used to prevent graft-versus-host disease. The influence of CSA on T-cell function has been extensively investigated; however, the effect of CSA on natural killer (NK) cells is less understood. NK cells were cultured with IL-2 and IL-15 with and without CSA for 1 week. Compared with controls, CSA-treated cultures showed fewer CD56 +CD16+KIR+ NK cells and a reciprocal increase in CD56+CD16-KIR- cells. These changes were due mainly to a reduced proliferation of the CD56dim NK-cell subpopulation and a relative resistance of CD56bright NK cells to CSA. Following coculture with K562 targets, CSAexposed NK cells differed from controls and lacked Ca2+ oscillations, nuclear factor of activated T cells (NFAT) dephosphorylation, and NFAT nuclear translocation. NK cells cultured in CSA retained cytotoxicity against K562, Raji, and KIR ligand-expressing lymphoblastoid cells. NK cells cultured in CSA showed increases in NKp30 and reductions in NKp44 and NKG2D. Following IL-12 and IL-18 stimulation, CSA-treated NK cells showed more IFN-γ-producing cells. Using in vitro NK-cell differentiation, progenitor cells gave rise to more CD56 +KIR- NK cells in the presence of CSA than controls. Collectively, these studies show that CSA influences NK-cell function and phenotype, which may have important implications for graft-versus-leukemia effects.