The inhibitory non-adrenergic non-cholinergic (i-NANC) innervation of the guinea pig airways was suggested to be mediated, at least partially, by nitric oxide (NO). The enzyme catalyzing the generation of NO and citrulline from L-arginine, nitric oxide synthase (NOS), was found to be identical with neuronal nicotinamide-adenine dinucleotide hydrogen phosphate (NADPH)-diaphorase. In the present study, we report the distribution of NOS in guinea pig lower airways and in vagal sensory and sympathetic ganglia as revealed by NOS immunohistochemistry and NADPH-diaphorase histochemistry. The distribution of NOS was identical using either technique and displayed a similar distribution pattern in all parts of the lower airways. Yet, the number of NOS-containing fibres was increasing from cervical trachea towards principal bronchi and decreasing to complete absence in bronchioli. Innervation with NOS-containing nerve fibres was densest in the smooth muscle layer and in the lamina propria of the mucosa. Single fibres were found in the respiratory epithelium. Labelling was absent from nerve fibres innervating the submucosal glands. Perivascular fibre networks enmeshed tracheal arteries, pulmonary arteries and veins. A substantial number of NOS-immunoreactive and NADPH-diaphorase-positive neurons was observed in vagal sensory ganglia, whereas such neurons were rather sparse in sympathetic ganglia. Tracheal and peribronchial ganglia of the airways were devoid of labelling. These findings suggest that extrinsic rather than intrinsic (tracheal and peribronchial) neurons are the source of NO release from guinea pig airway nerve fibres after electrical field stimulation. These extrinsic nerve fibres may originate from both sympathetic and vagal sensory ganglia.