This study sets out to compare the combinations of potential vasodilator transmitters expressed by sympathetic and pelvic vasodilator neurons of guinea-pigs. Triple-labelling fluorescence immunohistochemistry was used to examine immunoreactivity (IR) to vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS) and calcitonin gene-related peptide (CGRP) in lumbar sympathetic ganglia, and in perivascular axons supplying hindlimb skeletal muscles or pelvic viscera. Only 0.2% of VIP-IR nerve cell bodies in lumbar sympathetic ganglia (n = 4632 VIP-IR nerve cell profiles) contained NOS-IR, and one VIP-IR neuron contained CGRP-IR. The VIP-IR perivascular axons along the common and external iliac arteries, femoral artery and arteries to hindlimb muscles lacked NOS-IR and CGRP-IR. In contrast, all VIP-IR perivascular axons projecting from pelvic ganglia to the main uterine artery, and half of the VIP-IR axons along the internal iliac artery, contained NOS-IR and CGRP-IR. Thus, the neurochemical content of sympathetic vasodilator neurons to skeletal muscle arteries was clearly distinguishable from that of pelvic vasodilator neurons to the uterine vasculature. Furthermore, the autonomic dilation in each vascular bed is likely to be qualitatively different, and matched to the functional requirements of each target organ.