Glutamate is proved to be a neurotransmitter in the mammalian cochlea, transmitting signals between the inner hair cells and the afferent cochlear nerve terminals. The transmission in this synapse is modulated by the lateral olivocochlear efferent fibers by releasing dopamine and other neurotransmitters. This study undertakes to measure simultaneously the release of dopamine and glutamate from isolated guinea pig cochleae. We combined the in vitro microvolume superfusion method, that uses liquid scintillation analysis, to measure [3H]dopamine with high pressure liquid chromatography (HPLC) to determine the glutamate content of the superfusate at rest and during stimulation. The release of both neurotransmitters was significantly increased when electrical field stimulation was applied at a 10 Hz rate. The nonselective sodium-channel inhibitor tetrodotoxin (TTX) at 1 microM completely blocked the effect of stimulation, indicating the neural origin of both dopamine and glutamate. The dopamine receptor antagonist sulpiride at 100 microM and the dopamine receptor agonist bromocriptine at 20 microM did not change the release of glutamate. In contrast, both bromocriptine and sulpiride significantly increased the stimulation-evoked release of dopamine. The effect of sulpiride is most likely due to the blockade of dopamine autoreceptor. Possible explanations why bromocriptine increased the release include: (1) its partional agonist activity; (2) desensitizations of dopamine autoreceptors; or (3) the higher D1 receptor activity of bromocriptine than sulpiride. This study could provide further insights about the role of dopamine and glutamate in cochlear neurotransmission.