Nerve growth factor (NGF), a member of the neurotrophin family, enhances synthesis of neuropeptides in sensory and sympathetic neurons. The aim of this study was to examine the effect of NGF on airway responsiveness and determine if these effects are mediated through synthesis and release of substance P (SP) from the intrinsic airway neurons. Ferrets were instilled intratracheally with NGF or saline. Tracheal smooth muscle contractility to methacholine (MCh) and electrical field stimulation (EFS) was assessed in vitro. Contractions of isolated tracheal smooth muscle to EFS at 10 Hz and 30 Hz were significantly increased in the NGF treatment group (10Hz: 33.57±2.44%; 30Hz: 40.12±2.78%) compared to control group (10Hz: 27.24±2.14%; 30Hz: 33.33±2.31%). However, constrictive response to cholinergic agonist was not significantly altered the between the NGF treatment group and the control group. The NGF-induced modulation of airway smooth muscle to EFS was maintained in tracheal segments cultured for 24 h, a procedure that causes a significant anatomical and functional loss of SP-containing sensory fibers while maintaining viability of intrinsic airway neurons. The number of SP-containing neurons in longitudinal trunk and superficial muscular plexus and SP nerve fiber density in tracheal smooth muscle all increased significantly in cultured trachea treated with NGF. Pretreatment with CP-99994, an antagonist of neurokinin 1 (NK₁)-receptor, attenuated the NGF-induced increased contraction to EFS in cultured segments but had no effect in saline controls. These results show that the NGF-enhanced airway smooth muscle contractile responses to EFS are mediated by the actions of SP released from intrinsic airway neurons.