NO is known to reduce the hypoxia-induced increase in carotid body neural activity (CBNA). Acetylcholine (ACh), a known excitatory transmitter in the cat carotid body (CB), is released during hypoxia. This study addressed the impact of an NO precursor on ACh release during hypoxia. Both CBs from nine cats were prepared for incubation, then inserted into a medium and bubbled with three consecutive gas mixtures, hyperoxic, hypoxic, and a final hyperoxic mixture. This series of exposures was performed in the absence of L-arginine, followed by the three exposures in a 1mM L-arginine medium, and followed, thirdly, in a 10mM L-arginine medium. L-Arginine significantly attenuated the hypoxia-induced release of ACh. Two post-arginine procedures suggested strongly that the reduction in the ACh release was not due to a gradual exhaustion of carotid body ACh stores over the course of the experiment. The data are consistent with those reports showing that NO donors and precursors reduce the hypoxia-induced increase in CBNA, and further support a role for ACh in the hypoxia-induced increase in CBNA.