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The effects of putative efferent neurotransmitters on intracellular Ca2+ concentration ([Ca2+]i) in isolated vestibular hair cells (VHCs) of the guinea pig were determined using the Ca2+ sensitive dye Fura-2 and digital imaging microscopy. In the presence of 1 mM acetylcholine (ACh) there was a gradual increase in [Ca2+]i. In the presence of 10 μM ATP there was a rapid rise in [Ca2+]i. Thus, both ACh and ATP seem to be efferent neurotransmitters in VHCs of the guinea pig. When 100 μM Gamma-aminobutyric acid (GABA) was added there was a rapid increase in [Ca2+]i; thus GABA also seems to be an efferent neurotransmitter in VHCs. With the independent addition of 10 μM calcitonin gene-related peptide and 10 μM M- and L-enkephalin there were no significant increase in [Ca2+]i. We presume that these neuropeptides were functioning as efferent neuromodulators rather than as neurotransmitters.