Our purpose was to investigate the role of nitric oxide (NO) synthase substrate L-arginine (L-arg) in the regulation of airway ciliary beat frequency (CBF).We studied the effects of L-arg on CBF of rabbit tracheal epithelial cells by using high-speed digital microscopy and determined the expression and distribution of neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in intact tracheal mucosa and in isolated cilia by using immunohistochemistry and Western blot, respectively.L-arg dose- and time-dependently increased CBF compared with the control groups, and the effect was blocked by pretreatment with the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Immunoreactivity to 3 isoforms of NOS (nNOS, iNOS, and eNOS) was observed in cilia of epithelial cells, and the signal intensity of eNOS was stronger than that of both nNOS and iNOS. Western blot analysis of isolated ciliary preparations also detected all 3 isoforms of NOS, and the expression level is eNOS > nNOS > iNOS.L-arg increases the CBF of rabbit tracheal epithelial cells via the NOS-NO signal pathway.