COUP-TFs (chicken ovalbumin upstream promoter-transcription factors) are orphan members of the nuclear receptor superfamily of ligand-activated receptors for which their ligands have not been identified. The two mammalian proteins, COUP-TFI and COUP-TFII, share 80% sequence identity and regulate many aspects of mammalian development and differentiation. In this report, we systemically examined the temporal and spatial expression of COUP-TFI and COUP-TFII transcripts and, for the first time, their protein during development and functional maturation of the cochlea. Both COUP-TFI and COUP-TFII were expressed early in the developing otic vesicle. COUP-TFI expression correlated with the differentiation of hair cells and support cells in the organ of Corti, whereas COUP-TFII expression was down-regulated with differentiation of the organ of Corti. Furthermore, we report for the first time, that the generally nuclear COUP-TFI receptor protein was localized in the cytoplasm of maturing hair cells and pillar cells. Collectively, although COUP-TFI and COUP-TFII are homologues, the expression of each orphan receptor has a restricted and dynamic expression during cochlea development particularly during patterning and differentiation of the cochlear structures.