Presence of gentamicin (GM) in cochlear hair cells was detected by immunohistochemistry in guinea pigs (GPs) cochlea 1, 9 and 41 days after a 6-day treatment with GM at 60 mg/kg/day (s.c.). The number of GPs in each group was respectively 7, 12 and 6. Twelve other non-treated GPs served as controls. Cochlear function was measured, just before sacrifice, by VIIIth nerve compound action potential (CAP) audiograms. Functional and immunohistological evaluations were performed by two independent naïve observers respectively. Functional changes were minimal: only one out of the 25 treated GPs, from the 41-day group, showed significant threshold elevations on high frequencies. Meanwhile GM labelling was observed in most outer hair cells (OHCs) from the three rows of all the treated GPs, with radial and longitudinal gradients, and found similar in the 3 groups. These results 1) confirm that GM is significantly present in OHCs before the development of ototoxicity and 2) indicate that GM accumulates and is maintained inside the OHCs for very long periods of time, i.e. that its clearance from the hair cells, if any, would be very slow.