The distributions of nerve cells and fibres that are immunoreactive for nitric oxide synthase (NOS) have been investigated in the human gall-bladder. In addition, the colocalization of NOS immunoreactivity (IR) with neuropeptide Y (NPY), pituitary adenylyl cyclase activating peptide (PACAP), somatostatin (SOM), substance P (SP), tyrosine hydroxylase (TH) and vasoactive intestinal peptide (VIP)-IR was determined. Nitric oxide synthase-IR nerve cell bodies comprised 13 and 30% of nerve cells in ganglia of the fibromuscular and subepithelial layers, respectively. To determine these percentages, neuron-specific enolase-IR was used as a marker for all nerve cells. Although SOM- and VIP-IR nerve cell bodies were found in both ganglia, they rarely contained NOS-IR. In the fibromuscular layer, NOS-IR nerve fibres were abundant and most PACAP-, SOM- and VIP-IR fibres and many NPY-IR fibres were also NOS positive. No colocalization was observed between NOS- and SP- or TH-IR. In the mucosal layer, moderate numbers of NOS-IR fibres were found and the degree of colocalization of NOS-IR with each of NPY-, PACAP-, SOM-, SP- and VIP-IR were as follows: PACAP and NPY > VIP > SOM and SP. Nitric oxide synthase and TH were not colocalized in mucosal fibres. These results suggest that nerve fibres in the fibromuscular layer in the human gall-bladder with the chemical coding NOS/NPY/PACAP/SOM/VIP are axons of inhibitory motor neurons. Nitric oxide synthase-IR fibres in the mucosal layer that contained NPY, PACAP, SOM, SP and VIP with various degrees of colocalization probably contribute to the control of epithelial secretion or absorption.