Previous immunohistochemical and electrophysiological studies on various neurotransmitters revealed the tachykinin substance P (SP) as a neuromodulator in the auditory system of mammals. This study was performed in order to determine the immunohistochemical expression and distribution pattern of SP in the organ of Corti, especially in the inner (IHC) and outer hair cell (OHC) region of the guinea pig. We examined the immunoreactivity of SP of surface preparations by means of a fluorescence and a laser scanning microscope. The electrophysiological action of SP, N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were recorded at the subsynaptic afferent region of the IHCs using micro-iontophoretic techniques. The SP-like immunostaining could be detected at the basal and apical pole of the IHCs with a gradient distribution pattern from the basal to the apical turn. Furthermore, we could demonstrate SP-like immunoreactivity in OHCs with different occurrence in turns as well as in rows. Electrical activity was induced by applying SP, NMDA and AMPA perisynaptically to the IHCs. The selective SP antagonist spantide (D-Arg1, D-Trp7,9, Leu11-substance P) specifically blocked the SP-induced activity but without altering the activity of NMDA and AMPA. In contrast, specific NMDA or AMPA antagonists reversibly blocked either the NMDA- or AMPA-induced responses without affecting the SP-induced activity. These immunohistochemical and electrophysiological results confirm that SP may represent a neuromodulator function at the synapses of the IHCs in the guinea pig.