Atoh1 is a basic helix-loop-helix transcription factor that controls differentiation of hair cells (HCs) in the inner ear and its enhancer region has been used to create several HC-specific mouse lines. We generated a transgenic tetracycline-inducible mouse line (called Atoh1-rtTA) using the Atoh1 enhancer to drive expression of the reverse tetracycline transactivator (rtTA) protein and human placental alkaline phosphatase. Presence of the transgene was confirmed by alkaline phosphatase staining and rtTA activity was measured using two tetracycline operator (TetO) reporter alleles with doxycycline administered between postnatal days 0-3. This characterization of five founder lines demonstrated that Atoh1-rtTA is expressed in the majority of cochlear and utricular HCs. Although the tetracycline-inducible system is thought to produce transient changes in gene expression, reporter positive HCs were still observed at 6 weeks of age. To confirm that Atoh1-rtTA activity was specific to Atoh1-expressing cells, we also analyzed the cerebellum and found rtTA-driven reporter expression in cerebellar granule neuron precursor cells. The Atoh1-rtTA mouse line provides a powerful tool for the field and can be used in combination with other existing Cre recombinase mouse lines to manipulate expression of multiple genes at different times in the same animal.