Cochlear serotonergic innervation is constituted by efferent fibers projecting both to the area below the inner and the outer hair cells. Previous detection of serotonin (5-HT) metabolites and 5-HT receptor mRNAs suggests the existence of serotonergic synaptic activity in the cochlea. The present study explores this possibility through the effect of 6-nitroquipazine (6-NQ), a 5-HT selective reuptake inhibitor, on the basal turnover of 5-HT. The concentrations of 5-HT and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) were quantified by high performance liquid chromatography with electrochemical detection in blood-free cochleae of rats treated with 6-NQ or saline and kept under silent conditions. Treatment with 6-NQ induced a significant increase of the cochlear concentration of 5-HT and a significant reduction of 5-HIAA concentration with respect to saline treatment. These findings could indicate that 6-NQ induced the blockade of the 5-HT selective reuptake to the cochlear serotonergic fibers. This suggests that plasma membrane 5-HT transporters are present in cochlear serotonergic fibers. Even though the role of serotonergic innervation on cochlear physiology remains unknown, the existence of cochlear serotonergic synaptic activity is strongly supported by present contributions.