The patterns of colocalisation of neuropeptides, tyrosine hydroxylase (TH), and protein gene product 9.5 (PGP), were studied in nerve fibres supplying the upper and lower human ureter using a double labelling immunofluorescence technique. The majority (85%-95%) of nerve fibres within the ureter contained neuropeptide Y-like immunoreactivity (NPY-LIR), in combination with other peptides. Approximately 52%-63% of the total ureteral innervation was made up of NPY-LIR fibres also expressing TH-LIR, while 21%-42% of fibres contained NPY-LIR in combination with vasoactive intestinal polypeptide (VIP)-LIR. These two immunochemically defined classes did not overlap, since TH- and VIP-LIR were never present within the same nerve fibre. Other minor populations of neurones included those containing calcitonin gene-related peptide (CGRP)-LIR in combination with substance P (SP)-LIR (4%-17%) and those without SP (5%). Rare coexistences were also noted between CGRP- and VIP-LIR (1%-2%), CGRP- and NPY-LIR (< or = 1%), and CGRP- and TH-LIR (< 1%). Regional differences in innervation were found. There were fewer of each class of nerve fibres in the upper ureter compared to the lower ureter. In addition, the proportion of VIP/NPY-LIR fibres of the total innervation was less in the upper ureter, where they were very sparse. Differences in the distribution to various tissue targets were also observed. In the lower ureter, TH/NPY-LIR fibres were localised predominantly to the outer muscle fascicles and adventitia, while VIP/NPY immunoreactive nerves supplied the submucosa and inner smooth muscle fascicles. Both of these populations were also found around blood vessels. A population of presumptive sensory fibres expressing CGRP/SP-LIR were typically present immediately beneath the urinary epithelium and around blood vessels, and only very rarely within muscle fascicles. The finding that TH/NPY- and VIP/NPY-LIR fibres innervate different layers of the ureter raises the possibility that the muscle layers of the ureter may be independently controlled.