Cochlear outer hair cells (OHCs) are dominantly innervated by efferents, with acetylcholine (ACh) being their principal neurotransmitter. ACh activation of the cholinergic receptors on isolated OHCs induces calcium influx through the ionotropic receptors, followed by a large outward K+ current through nearby Ca2+-activated K+ channels. The outward K+ current hyperpolarizes the cell, resulting in the fast inhibitory effects of efferent action. Although the ACh receptors (AChRs) in adult OHCs have been identified and the ACh-induced current responses have been characterized, it is unclear when the ACh-induced current responses occur during development. In this study we attempt to address this question by determining the time of onset of the ACh-induced currents in neonatal gerbil OHCs, using whole cell patch-clamp techniques. Developing gerbils ranging in age from 4 to 12 days were used in these experiments, because efferent synaptogenesis and functional maturation of OHCs occur after birth. Results show that the first detectable ACh-induced current occurred at 6 days after birth (DAB) in 12% of the basal turn cells with a small outward current. The fraction of responsive cells and the size of outward currents increased as development progressed. By 11 DAB, the fraction of responsive cells and the current size were comparable with those of adult OHCs. The results indicate that the maturation of the ACh-induced response begins around 6 DAB. It appears that the development of ACh-induced responses occur during the same time period when OHCs develop motility but before the onset of auditory function, which is around 12 DAB when cochlear microphonic potentials can first be evoked with acoustic stimulation in gerbils.