Sensitivity of the electromotility of isolated guinea pig outer hair cells (OHCs) to acetylcholine (ACh) was examined. OHCs were held in a partitioning microchamber in a position so that their ciliary poles were inserted and their somatic length changes measured. Transcellular square-wave stimuli were delivered to record voltage-to-movement conversion of the cells. ACh was applied to the synaptic poles. The transfer function of electromotility intimated shifts of the membrane potential with concomitant gain changes: gain decreases when the membrane potential is shifted in hyperpolarization direction and gain increases when the membrane potential is shifted in the opposite direction due to application of ACh in 100-500 microM concentration. Clear gain increase of the electromotility in basal turn OHCs to ACh was observed, whereas inconsistent results for apical turn OHCs were found. The latter is probably due to the pharmacological dose of ACh used. This possibility is further supported by results in which ACh abolished the sensitivity of the magnitude response of electromotility to DC depolarizing bias.