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Detailed understanding of neurofilament protein distribution in the inner ear can shed light on regulatory mechanisms involved in neuronal development of this tissue. We assessed the spatio-temporal changes in the distribution of neurofilaments in the developing rat inner ear between embryonic day 12 and 30 days after birth, using antibodies against phosphorylated as well as non-phosphorylated light (NFL), medium (NFM) and heavy (NFH) neurofilament subunits. Our results show that during development, the onset of neurofilament expression in the rat inner ear is on embryonic day 12, earlier than previously shown. We demonstrate that neurofilament subunits of different molecular weight emerge in a developmental stage-dependent order. In addition, we determined that neurofilaments of the vestibular nerve mature earlier than neurofilaments of the cochlear nerve. Cochlear neurofilament maturation progresses in a gradient from base to apex, and from inner to outer hair cells. The sequential pattern of neurofilament expression we describe may help understand the consequences of certain mutations, and contribute to develop therapeutic strategies.