Vasoactive intestinal peptide (VIP) and nitric oxide (NO) are potent mediators of neural airway smooth muscle relaxation. The major contractile mediator released by airway nerves under physiological conditions is acetylcholine (ACh). In the present study, we have correlated the immunohistochemical distribution of the relaxant mediators using antisera to VIP, to the marker enzyme of catecholamine synthesis tyrosine hydroxylase (TH) and to the NO-generating enzyme NO-synthase (NOS) with the distribution of the ACh-synthesizing enzyme, choline acetyltransferase (ChAT), and of substance P (SP), a neuropeptide present in sensory nerve fibers. In guinea-pig airways, VIP-and NOS-immunoreactivity (IR) were present in numerous nerve fibers in the airway smooth muscle and around submucosal glands; some fibers were also seen in the lamina propria and around blood vessels. The neuronal cell bodies in the intrinsic ganglia were devoid of both VIP-and NOS-IR. In contrast, all neuronal cell bodies in the intrinsic ganglia were immunoreactive for ChAT. In human airways, immunoreactivity for VIP, NOS, and ChAT was found in airway intrinsic neuronal perikarya. Whereas ChAT-IR appeared to be most frequent in the cell bodies, VIP-IR was seen in the largest number of nerve fibers in the airways. Therefore, in guinea pigs, a clear neuroanatomical and neurochemical separation of relaxant and of constrictor pathways is seen, whereas in human airways, both separate pathways as well as coexpression of VIP-/NOS-and of ChAT-IR are found.