The neuropeptide substance P (SP), as a major mediator of neuroimmunomodulatory activity, modulates diverse functions of immune cells including macrophages. In the current study, we focus on the yet uncertain role of SP in enhancing the inducible/inflammatory chemokine response of macrophages and the signaling mechanism involved. The effect is studied on the murine macrophage cell line RAW 264.7 as well as isolated primary macrophages. Our data show that SP, at nanomolar concentrations, elicits selective chemokine production from murine macrophages. Among the chemokines examined, MIP-2 and MCP-1 are two major chemokines synthesized by macrophages in response to SP. Furthermore, SP treatment strongly induces the classical pathway of IB-dependent NF-B activation and enhances DNA-binding as well as transactivation activity of the transcription factor. SP-evoked transcriptional induction of chemokines is specific, since it is blocked by treatment with selective neurokinin-1 receptor antagonists. Moreover, SP stimulation of macrophages activates the ERK1/2 and p38 MAPK, but not JNKs. Blockade of these two MAPK pathways with specific inhibitors abolishes SP-elicited nuclear translocation of phosphorylated NF-B p65 and NF-B-driven chemokine production, suggesting that the two MAPKs lie in the signaling pathways leading to the chemokine response. Collectively, our data demonstrate that SP enhances selective inflammatory chemokine production by murine macrophages via ERK/p38 MAPK-mediated NF-B activation.