Steroids have been shown to inhibit the function of fresh or IL-2-activated natural killer (NK) cells. Since IL-15 plays a key role in NK-cell development and function, we comparatively analyzed the effects of methylprednisolone on IL-2- or IL-15-cultured NK cells. Methylprednisolone inhibited the surface expression of the major activating receptors NKp30 and NKp44 in both conditions, whereas NK-cell proliferation and survival were sharply impaired only in IL-2-cultured NK cells. Accordingly, methylprednisolone inhibited Tyr phosphorylation of STAT1, STAT3, and STAT5 in IL-2-cultured NK cells but only marginally in IL-15-cultured NK cells, whereas JAK3 was inhibited under both conditions. Also, the NK cytotoxicity was similarly impaired in IL-2- or IL-15-cultured NK cells. This effect strictly correlated with the inhibition of ERK1/2 Tyr phosphorylation, perforin release, and cytotoxicity in a redirected killing assay against the FcRgamma(+) P815 target cells upon cross-linking of NKp46, NKG2D, or 2B4 receptors. In contrast, in the case of CD16, inhibition of ERK1/2 Tyr phosphorylation, perforin release, and cytotoxicity were not impaired. Our study suggests a different ability of IL-15-cultured NK cells to survive to steroid treatment, thus offering interesting clues for a correct NK-cell cytokine conditioning in adoptive immunotherapy.