The release of endogenous neurotransmitters plays an important role in the airway mucosal defense system. We studied the in vitro effect of methacholine, a β-methyl ester of acetylcholine, on the ciliary beat frequency (CBF) of human adenoid explants and its mechanism of action. Tissue explants were cultured at 35°C and covered with 1.0 mL of culture medium: minimum essential Eagle's medium (MEM) containing L-arginine (1.2 × 10⁻³ mol/L). Methacholine was added to the cultured tissue at concentrations of 10⁻¹⁰, 10⁻⁸, and 10⁻⁶ mol/L. The CBF was determined by phase contrast microscopy and microphotometry. Methacholine increased CBF in a dose-dependent manner with a maximum increase of 23.0% ± 1.8% (p <.001). Atropine (10⁻⁶ mol/L) significantly inhibited the ciliostimulatory effects of methacholine (p <.0007). The role of endogenous prostaglandins in methacholine-induced ciliostimulation was determined by treating specimens with a cyclooxygenase inhibitor (diclofenac sodium). Diclofenac (10⁻⁶ mol/L) significantly inhibited the ciliostimulatory effects of methacholine (p <.0007). To determine if nitric oxide (NO) acts as an intermediary in ciliostimulation by methacholine, endogenous NO production was inhibited by treating specimens with an L-arginine analog, N^G-nitro-L-arginine methyl ester (L-NAME), prior to addition of methacholine. L-NAME (10⁻⁶ mol/L) inhibited the effects of methacholine in L-arginine-free MEM (p <.008), and this inhibition was reversed by L-arginine (10⁻³ mol/L). To further examine the actions of NO in methacholine-induced ciliostimulation, a cyclic guanosine 3′5′-monophosphate (cGMP) kinase inhibitor (KT-5823) was used, prior to the addition of methacholine. KT-5823 (10⁻⁶ mol/L) significantly inhibited the effects of methacholine (p <.0001). Ciliostimulation by methacholine in human upper airway mucosa involves both prostaglandin and NO second messengers and activation of a cGMP-dependent kinase.