The extrinsic efferent innervation of the distal colon and rectum of the guinea pig was compared, by using retrograde tracing combined with immunohistochemistry. Application of the carbocyanine tracer DiI to the rectum filled significantly greater numbers of extrinsic neurons than similar injections into the distal colon. Approximately three-fourths of all filled neurons from either location were either sympathetic or parasympathetic; the rest were spinal sensory neurons. Nerve cell bodies in sympathetic prevertebral ganglia labelled from the two regions were similar in number. Both regions were innervated by sympathetic neurons in paravertebral ganglia; however, the rectum received much more input from this source than the colon. The rectum received significantly more input from pelvic ganglia than the colon. The rectum also received direct innervation from two groups of neurons in the spinal cord. Neurons located in the spinal parasympathetic nucleus in segment S2 and S3 were labelled by DiI injected into the rectal wall. Similar numbers of neurons, located in intermediolateral cell column and dorsal commissural nucleus of lumbar segments, also projected directly to rectum, but not colon. The great majority (>80%) of retrogradely labelled nerve cell bodies in sympathetic ganglia were immunoreactive for tyrosine hydroxylase. In pelvic ganglia, retrogradely labelled neurons contained choline acetyltransferase and/or nitric oxide synthase or tyrosine hydroxylase. Although the rectum and colon in this species are continuous and macroscopically indistinguishable, they have significantly different patterns of extrinsic efferent innervation, presumably reflecting their different functions.