Three series of double-labeling experiments were carried out in a study of the collateralization of brainstem nuclei which project to the spinal cord in the rat. The fluorescent tracer Diamidino Yellow Dihydrochloride (DY X 2HCl) was injected in one half of the spinal gray and white matter at T7-T8 or T13-L1. Subsequently, either True Blue (TB), wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) or free HRP were injected ipsilaterally in the gray matter at C5-C8. The distributions of single and double retrogradely labeled neurons were studied in the following cell groups: red nucleus, interstitial nucleus of Cajal, ventrolateral pontine tegmentum, nuclei coeruleus and subcoeruleus and nuclei raphe magnus and raphe pallidus including the adjoining ventral reticular formation. The numbers of TB- or HRP-labeled neurons present in those nuclei were counted and the percentages of double-labeled neurons were calculated when TB or HRP had been used in combination with DY X 2HCl. The results indicate: The HRP-TMB reaction product and the DY X 2HCl fluorescence can be visualized simultaneously in retrogradely single- or double-labeled neurons. The distributions of single- and double-labeled neurons in the various brainstem nuclei were entirely comparable when using TB with DY X 2HCl or HRP with DY X 2HCl. The percentages of double-labeled neurons obtained with HRP and DY X 2HCl were consistent over a series of cases, and were comparable to those obtained with TB and DY X 2HCl in several structures. However, in the red nucleus slightly lower percentages of double-labeled neurons were obtained using HRP and DY X 2HCl as compared with the percentages obtained using TB and DY X 2HCl.