The neuropeptide substance P (SP) is one of the principal mediators of neurogenic inflammation as well as a neurotransmitter in nociceptive affect neurons. The mechanisms by which binding of SP to its receptor stimulates diverse downstream biologic effects remain unknown. In order to elucidate this process we have established stably transfected cell lines expressing functional rat SP receptors (KNRK-SPR). When stimulated by SP, KNRK-SPR cells respond by simultaneously mobilizing intracellular Ca2+ and increasing cAMP levels. To determine if SP stimulation activates downstream transcriptional regulatory factors, we transfected KNRK-SPR cells with plasmids containing the activator protein 1 (AP-1) and cAMP-responsive (CRE) enhancer elements coupled to the chloramphenicol acetyltransferase (CAT) reporter gene. Stimulation with SP 1–1,000 nM caused a 1.5-to 2-fold increase in CAT activity in both AP-1-CAT- and CRE-CAT-transfected KNRK-SPR cells. Northern and Western blot analyses demonstrate that the mechanism by which SP stimulates AP-1 enhancer activity involves increases in both c-jun mRNA and protein. Moreover, gel retardation assays with oligomers containing the AP-1 and CRE binding sites showed that SP induces specific retardation bands consistent with increases in AP-1 and CRE complexes. These experiments suggest that SP-mediated stimulation of cells involves the participation of two signaling pathways resulting in several transcriptional regulatory mechanisms being activated.