We have recently reported that interleukin (IL)-15 and IL-2, which signal through IL-2Rbetagamma, oppositely regulate expression of the proinflammatory chemokine receptor CX3CR1. Here we delineate molecular mechanisms responsible for this paradox. By using a luciferase reporter plasmid, we identified a 433-bp region spanning the major transcriptional start point of human CX3CR1 that, when expressed in human peripheral blood mononuclear cells (PBMCs), possessed strong constitutive promoter activity. IL-2 and IL-15 treatment increased and abolished this activity, respectively, mimicking their effects on endogenous CX3CR1. IL-2 and IL-15 have been reported to also have opposite effects on the immunoregulatory transcription factor NFAT (nuclear factor of activated T cells), and the 433-bp region contains a kappaB-like NFAT site. The effects of IL-15 and IL-2 on both CX3CR1 reporter activity and endogenous CX3CR1 transcription in PBMCs were abolished by the NFAT inhibitors cyclosporin A and VIVIT. Moreover, mutation of the kappaB-like NFAT sequence markedly attenuated IL-2 and IL-15 modulation of CX3CR1 promoter-reporter activity in PBMCs. Furthermore, chromatin immunoprecipitation revealed that IL-15 promoted specific recruitment of NFAT1 but not NFAT2 to the CX3CR1 promoter, whereas IL-2 had the converse effect. This appears to be relevant in vivo because mouse CX3CR1 mRNA was expressed in both PBMCs and splenocytes from NFAT1-/- mice injected with recombinant IL-15 but was undetectable in cells from IL-15-injected NFAT1+/+ BALB/c mice; as predicted, IL-2 up-regulated cx3cr1 in both mouse strains to a similar extent. Thus, by pharmacologic, genetic, and biochemical criteria in vitro and in vivo, our results suggest that IL-15 and IL-2 oppositely regulate CX3CR1 gene expression by differentially recruiting NFAT1 and NFAT2 to a kappaB-like NFAT site within the CX3CR1 promoter. We propose that expression of CX3CR1 and possibly other immunoregulatory genes may be determined in part by the balance of NFAT1 and NFAT2 activity in leukocytes.