Airway hyperresponsiveness (AHR) is present in almost all patients with symptomatic asthma, yet its mechanism is not well understood. Airway inflammation is thought to be an important underlying mechanism involved in causing AHR. Recent studies indicate release of neuropeptides from C-fiber endings plays a pivotal role in airway inflammation. Substance P (SP) is a critical neurotransmitter of sensory C-fiber and a well-known effector of inflammatory response. However, roles of other neuropeptides and interaction among these neuropeptides in airway inflammation and AHR were largely unknown. Calcitonin gene-related peptide (CGRP), another intrapulmonary neuropeptide that functions as a potent vasodilator and neutrophils activator, is released from the same C-fiber ending as SP is released. By using an ozone-stressing animal model, previously we had demonstrated that CGRP might be involved in the development of AHR in rabbits. To extend the functional roles of SP, and to explore the possible interactive roles of SP and CGRP in airway inflammation, we examined expressions of SP, SP receptor (neurokinin 1, or NK-1R) and CGRP in vivo and ex vivo. We exposed guinea pigs at intervals to inhalation of ozone to induce airway inflammation. Animals were sacrificed at different time points; SP, SP receptor and CGRP expression were determined during the onset and progression of airway inflammation by radioimmunoassay, immunohistochemistry and in situ hybridization. Our data showed that after exposure to ozone, the concentration of SP in lung homogenate and the number of SP-immunoreactive cell bodies in lung slides increased within 24h, peaked on day 2, and then decreased slowly. Interestingly, CGRP expressions exhibited a similar temporal and spatial pattern, and there was a strong correlation between SP expression and CGRP expression, indicating a possible cooperative action of these two neuropeptides. We also noted an increased expression of SP receptor NK-1R in the development of airway inflammation. In order to test the hypothesis that CGRP as a coexisting neurotransmitter with SP can regulate the expression of NK-1R in the lung, and contribute to the SP-mediated inflammatory response, we used in vitro lung tissue culture to determine the effect of CGRP on NK-1R expression. We found that NK-1R expression was induced by CGRP incubation at both mRNA and protein levels, and the induction was attenuated by additions of the inhibitors of Protein Kinase A (PKA) pathway, Calmodulin-dependent Kinase pathway, and Tyrosine Protein Kinase pathway. In conclusion, our data provide compelling evidence that SP and CGRP are involved in the development of airway inflammation. The interaction between SP and CGRP is likely to contribute to the pathogenesis of AHR and other lung inflammatory diseases.