Mucociliary activity is an important clearance mechanism in the respiratory system of air breathing vertebrates. Substance P (SP) and acetylcholine play an important role in the stimulation of the mucociliary transport in the frog palate. In this study, anterograde neuronal tracing was combined with immunocytochemistry for NK-1R (SP receptor) and Muc3R (acetylcholine receptor) in the palate. The nerve fibers in the palate were labeled with the anterograde tracer Biotinylated Dextran Amine (BDA). The optimal labeling of BDA fibers in the palate was obtained at 8 days of exposure. Immunoflorecence shows that NK-1R and Muc3R are co-localized in the membranes of epithelial and goblet cells in the palate. Molecular characterization of SP receptor was performed by isolation of RNA from brain and palate followed by cDNA synthesis. Ultrastructural study of the palate showed axonal-like endings with vesicles in connection with epithelial and goblet cells. These results further support the concerted action of both neurotransmitters in the regulation of mucociliary activity in the frog palate.