It has been shown that inhaled cigarette smoke activates pulmonary C-fibers and rapidly adapting receptors in the airways, and that nicotine contained in the smoke is primarily responsible. This study was carried out to determine whether nicotine alone can activate pulmonary sensory neurons isolated from rat vagal ganglia; the response of these neurons was determined by fura-2-based ratiometric Ca²⁺ imaging. Our results showed: 1) Nicotine (10^-4 M, 20 s) evoked a transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in 175 of the 522 neurons tested (&#8710; [Ca²⁺]_i = 142.2 ± 12.3 nM); the response was reproducible, with a small reduction in the peak amplitude, in the same neurons when the challenge was repeated 20 min later. 2) A majority (59.7%) of these nicotine-sensitive neurons were also activated by capsaicin (10^-7 M). 3) 1,1-dimethyl-4-phenylpiperazinium (DMPP; 10^-4 M, 20 s), a selective agonist of the neuronal nicotinic acetylcholine receptors (NnAChRs), evoked a similar pattern of response as that of nicotine in these neurons. 4) The responses to nicotine and DMPP were either totally abrogated or markedly attenuated by pretreatment with hexamethonium (10^-4 M). 5) In anesthetized rats, a right-atrial bolus injection of nicotine (75-200 µg/kg) evoked an immediate (latency < 1-2 s) and intense burst of discharge in 47.8% of the pulmonary C-fiber endings and 28.6% of the RARs tested. In conclusion, nicotine exerts a direct stimulatory effect on vagal pulmonary sensory neurons, and the effect is probably mediated through an activation of the NnAChRs expressed on the membrane of these neurons.