After injecting Diamidino yellow (DY) and Fast blue (FB), respectively, into the trigeminal sensory complex and lumbar cord segments, neurons were doubly labeled retrogradely with both DY and FB in the periaqueductal gray (PAG) and dorsal raphe nucleus (DR). The fluorescent retrograde double-labeling method combined with serotonin (5HT) immunofluorescence histochemistry revealed that more than 70% of the doubly labeled PAG/DR neurons showed 5HT-like immunoreactivity (5HT-LI). These 5HT-LI PAG/DR neurons may modulate nociceptors in the trigeminal sensory complex and spinal cord by sending axon collaterals to these regions.