We have employed immunohistochemistry and the retrograde transport of Fast blue to study the origin of serotoninergic projections to the lumbosacral spinal cord at different stages of development in the North American opossum. A few serotoninergic axons are present in the lumbosacral cord at birth, 12 days after conception, and serotoninergic neurons are numerous in the brainstem where they are present in most, if not all, of the areas which contain them in the adult animals. A few neurons of the caudal raphe and adjacent reticular formation were labeled by lumbar injections of Fast blue on postnatal day 1, and by postnatal day 3, labeled neurons were numerous within all areas which provide serotoninergic projections to the lumbosacral cord in adult animals. By postnatal day 11, it was possible to combine Fast blue labeling with immunofluorescence to show that some of the labeled neurons were serotoninergic. By postnatal day 24, neurons which provide serotoninergic projections to the lumbosacral cord were especially numerous and some of them were found in areas which do not provide comparable projections in adult animals. In developing and adult animals, few, if any, neurons were labeled in the dorsal raphe or superior central nuclei. We have shown previously that serotoninergic axons do not innervate laminae I and II of the lumbosacral cord until approximately postnatal day 50, although they are present in the marginal zone at birth and have grown into laminae III-X by postnatal day 15. Since serotoninergic axons which project to laminae I and II originate within the raphe magnus and adjacent reticular formation, and those areas provide serotoninergic projections to the spinal cord well before postnatal day 50, it is possible that serotoninergic innervation of laminae I and II is provided by late growth of collaterals from axons that have been present in the marginal zone for some time.