Recent studies have demonstrated the use of manganese ion (Mn2+)) as an in vivo neuronal tract tracer. In contrast to histological approaches, manganese tracing can be performed repeatedly on the same living animal. In this study, we describe the neuroaxonal tracing of the auditory pathway in the living guinea pig, relying on the fact that Mn2+ ion enters excitable cells through voltage-gated calcium channels and is an excellent MRI paramagnetic tract-tracing agent. Small focal injections of Mn2+ ion into the cochlea produced significant contrast enhancement along the known neuronal circuitry. This in vivo approach, allowing repeated measures, is expected to open new vistas to study auditory physiology and to provide new insights on in vivo axonal transport and neuronal activity in the central auditory system.