Location of the lung respiratory rhythm generator (RRG) in the bullfrog brainstem was investigated by examining neurokinin-1 and µ-opioid receptor (NK1R, µOR) colocalization by immunohistochemistry and characterizing the role of these receptors in lung rhythm and episodic pattern generation. NK1R and µOR occurred in brainstems from all developmental stages. In juvenile bullfrogs a distinct area of colocalization was coincident with high-intensity fluorescent labeling of µOR; high-intensity labeling of µOR was not distinctly and consistently localized in tadpole brainstems. NK1R labeling intensity did not change with development. Similarity in colocalization is consistent with similarity in responses to substance P (SP, NK1R agonist) and DAMGO (µOR agonist) when bath applied to bullfrog brainstems of different developmental stages. In early stage tadpoles and juvenile bullfrogs, SP increased and DAMGO decreased lung burst frequency. In juvenile bullfrogs, SP increased lung burst frequency, episode frequency, but decreased number of lung bursts per episode and lung burst duration. In contrast, DAMGO decreased lung burst frequency and burst cycle frequency, episode frequency, and number of lung bursts per episode but increased all other lung burst parameters. Based on these results, we hypothesize that NK1R and µOR colocalization together with a metamorphosis-related increase in µOR intensity marks the location of the lung RRG but not necessarily the lung episodic pattern generator.