The mouse Nkx-5.1 and Nkx-5.2 genes have been identified by sequence homology to Drosophila NK genes within the homeobox domain. Here, we report the isolation of the Nkx-5.2 cDNA and a detailed comparative analysis of the spatio-temporal expression patterns for Nkx-5.1 and Nkx-5.2 genes. Nkx-5.2 transcripts are first detected in E13.5 embryos where they colocalize with Nkx-5.1 mRNA in the developing central nervous system and the inner ear. However, the onset of Nkx-5.1 transcription begins much earlier in 10 somite stage embryos (E8.5) in the otic placode and the branchial region. Nkx-5.1 expression in the ear persists until birth, whereas in branchial arches it is transient between E8.5 to E11.5. Transcript distribution appears regionalized in the otic vesicle concentrating at the anterior and posterior margin and later at the dorsal side of the otocyst. These domains are distinct from regions expressing Pax-2 and sek, two other early markers for otic development. From E11.5 to birth several Nkx-5.1 expression domains appear in the brain between the ventral diencephalon and the myelencephalon. The same expression domains also exist for Nkx-5.2 beginning at E13.5. The regionally restricted expression pattern of both Nkx-5 genes during mouse development suggests their involvement in cell type specification of neuronal cells.