Using flow cytometric and RNase protection assays, this study examined the expression of chemokine receptors in nonactivated natural killer (NK) cells and compared this expression with NK cells activated with interleukin (IL)-2, which either adhered to plastic flasks (AD) or did not adhere (NA). None of the NK cell subsets expressed CXCR2, CXCR5, or CCR5. The major differences between these cells include increased expression of CXCR1, CCR1, CCR2, CCR4, CCR8, and CX₃CR1 in AD when compared to NA or nonactivated NK cells. The chemotactic response to the CXC and CC chemokines correlated with the receptor expression except that all 3 populations responded to GRO-α, despite their lack of CXCR2 expression. Pretreatment of these cells with anti-CXCR2 did not inhibit the chemotactic response to GRO-α. In addition, nonactivated and NA cells responded to fractalkine, although they lack the expression of CX₃CR1. This activity was not inhibited by anti-CX₃CR1. Viral macrophage inflammatory protein (vMIP)-I, I-309, and TARC competed with the binding of ¹²⁵I-309 to AD cells with varying affinities. Transforming growth factor (TGF)-β1 but not any other cytokine or chemokine examined including interferon (IFN)-γ, MIP-3β, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC) or I-309, up-regulated the expression of CXCR3 and CXCR4 on NK cell surface. This is correlated with increased chemotaxis of NK cells treated with TGF-β1 toward stromal cell–derived factor (SDF)-1α and interferon-inducible protein-10 (IP-10). Messenger RNA for lymphotactin, RANTES, MIP-1α, and MIP-1β, but not IP-10, monocyte chemotactic protein (MCP)-1, IL-8, or I-309 was expressed in all 3 NK cell subsets. Our results may have implications for the dissemination of NK cells at the sites of tumor growth or viral replication.