In the mammalian inner ear, outer hair cells (OHCs) control mechanics of the cochlear partition. They do so through their electromotility, a property where cells generate longitudinal forces exerted by a motor protein named prestin (SLC26A5), in the cells lateral membrane. It has been proposed that prestin acts as an incomplete anion transporter (Oliver et al, 2001). To study this possibility further we have investigated how OHC currents respond to anion replacement. We used a novel preparation in which coils of the organ of Corti (approximately half a turn) were imaged without further dissociation. Coils were dissected from the cochlea of adult guinea-pigs humanely killed according to a Schedule 1 protocol. The coils were dissected on ice and held by a fine mesh grid on the stage of an upright microscope under temperature control. Identified OHCs were recorded under tight-seal whole-cell conditions for greater than 40 minutes with little deterioration. The recording patch pipettes containing (in mM): K, 140, EGTA 0.5; Mg 2; Cl 144; Hepes 10; (adjusted to pH 7.3 and 320 mOsM). The extracellular solution contained: Na 140; K 4; Cl 150; Ca 1; Mg 2; Hepes 10, pH 7.3 and matching osmotic strength. Where necessary chloride was replaced with gluconate and solutions were exchanged by continuous perfusion of the chamber. Cells from the cochlear apical turn exhibited a nonlinear I-V curve with outward rectification prominent above -20mV. The OHC input slope conductance at -50 mV was 10.3 ± 1.8 nS (s.e.m., n=16; range 1.6-16.6 nS) whereas at +30 mV the slope increased by a mean factor of 2.12. Reduction of external chloride reduced the outward current at +30mV by approximately 50%. Application of 1 mM 4-AP, proposed to block IK in apical OHCs (Mammano & Ashmore, 1996), did not remove the effect of low external chloride. A tail current analysis suggested that outward current at depolarised levels was a a result of a significant chloride movement through the membrane although the replacing anion, gluconate, was partially permeable. A similar permeability was found for sulfamate. Lowering intracellular chloride to 30 mM and to 1 mM in the pipette reduced outward current, the effect of then lowering external chloride remaining in accordance with modified GHK equation predictions. The simplest model which explains the present data is one where significant chloride movement occurs through prestin, a transporter with a chloride-activated gate at both external and cytoplasmic sites. The high density of prestin (>10⁷ copies / cell) in the OHC lateral membrane provides an opportunity to measure these currents.