A previous study showed that class III โค-tubulin, a widely used neuron-specific marker, is expressed in mature and regenerating hair cells but not the support cells of the avian inner ear. We investigated the expression of this marker in the developing avian inner ear. We found that class III โค-tubulin is not neuron-specific in the avian embryo, but appears to accumulate in neuronal cell types, including hair cells, about the time of their differentiation. In the developing inner ear, some degree of class III โค-tubulin immunoreactivity is found in all regions of the otic epithelium from its formation as the otic placode (stage 10 [embryonic day, E1.5]) until about stage 21 (E3.5), when the prospective tegmentum vasculosum begins to lose its staining. By stage 35 (E8-9), most of the nonsensory epithelia have lost their class III โค-tubulin staining, leaving distinct regions of staining between the morphological compartments of the inner ear. Concurrent with the loss of staining from nonsensory regions, the hair cells of the sensory epithelia accumulate class III โค-tubulin, whereas the supporting cells decrease their staining. We also observed a similar pattern of development in another hair cell organ, the paratympanic organ. Double labeling with the 275 kD hair cell antigen (HCA) indicated that the majority of hair cells identifiable with class III โค-tubulin are HCA-positive. Additionally, presumptive hair cells were identified which were not within defined sensory epithelia. Our findings show that class III โค-tubulin can be used as an early marker for hair cell differentiation in all hair cell sensory epithelia in the chicken.