Neurons in the developing auditory system fire bursts of action potentials before the onset of hearing. This spontaneous activity promotes the survival and maturation of auditory neurons and the refinement of synaptic connections in auditory nuclei; however, the mechanisms responsible for initiating this activity remain uncertain. Previous studies indicate that inner supporting cells (ISCs) in the developing cochlea periodically release ATP, which depolarizes inner hair cells (IHCs), leading to bursts of action potentials in postsynaptic spiral ganglion neurons (SGNs). To determine when purinergic signaling appears in the developing cochlea and whether it is responsible for initiating auditory neuron activity throughout the prehearing period, we examined spontaneous activity from ISCs, IHCs, and SGNs in cochleae acutely isolated from rats during the first three postnatal weeks. We found that ATP was released from ISCs within the cochlea from birth until the onset of hearing, which led to periodic inward currents, Ca(2+) transients, and morphological changes in these supporting cells. This spontaneous release of ATP also depolarized IHCs and triggered bursts of action potentials in SGNs for most of the postnatal prehearing period, beginning a few days after birth as IHCs became responsive to ATP, until the onset of hearing when ATP was no longer released from ISCs. When IHCs were not subject to purinergic excitation, SGNs exhibited little or no activity. These results suggest that supporting cells in the cochlea provide the primary excitatory stimulus responsible for initiating bursts of action potentials in auditory nerve fibers before the onset of hearing.