The distribution and patterns of colocalization of nitric oxide synthase (NOS), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY) and the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) were examined in nerve fibers supplying the human lower ureter using double label immunofluorescence. Many nerve fibers immunoreactive for NOS were observed within the ureter. Positive varicose fibers were seen running longitudinally within the smooth muscle bundles, particularly those of the inner layers of the ureter. Immunoreactive axons were also prominent within the subepithelium, and as plexi surrounding many blood vessels. The colocalization studies indicated that NOS was never present in presumptive sympathetic nerve fibers expressing TH. All fibers containing VIP, however, were also immunoreactive for NOS. In addition, a minor population of NOS fibers did not contain VIP. Neuropeptide Y coexisted with NOS in a significant number of nerve terminals, although fibers expressing only NPY were equally common. Several immunochemically distinct nerve populations can therefore be distinguished in the human ureter: (1) nerves containing NOS either with or without VIP; (2) NOS-immunoreactive fibers with NPY; and (3) those fibers expressing TH or NPY which do not contain NOS. The results indicate that some non-noradrenergic peptide-containing nerves in the human ureter have the capacity to synthesize nitric oxide (NO), and that NO may be involved in the regulation of ureteric motility.