The use of fluorescent dyes has been a major improvement for paths tracing studies. However, these tracers present different properties and have to be chosen carefully. The present study compares the ability of different tracers to specifically label phrenic motoneurons (PMNs) innervating the rat diaphragm. The administration of fluorogold (FG) from the transected phrenic nerve specifically labeled PMNs in the ipsilateral spinal cord. However, when FG was injected into one hemidiaphragm, in addition with ipsilateral PMNs, a less intense artifactual labeling was observed in the spinal cord (mainly in contralateral PMNs) and in the medulla oblongata (mainly in the area postrema and cranial motor nuclei). Similar results were observed using horseradish peroxidase, while no labeling was observed after injection of nuclear yellow or diamidino yellow into the diaphragm. By contrast, the dextran amine fluororuby (FR) and the carbocyanine DiAsp selectively and exclusively labeled ipsilateral PMNs 2 or 3 weeks after injection into the diaphragm, respectively. The lipophilic properties of DiAsp and the high molecular weight of FR may prevent their diffusion to adjacent tissues and into the blood stream which seems to account for the artifactual labeling observed with the other tracers. The higher homogeneity and quality of the labeling observed with FR compared to DiAsp make it the most appropriate tracer for the specific monosynaptic fluorescent labeling of PMNs after injection into the diaphragm.