A mouse homeobox gene, Nkx-1.2 , (previously termed Sax-1) that is closely related to the Drosophila NK-1/S59 gene was cloned, and genomic DNA and cDNA were sequenced. Nine Nkx-1.2 cDNA clones were found that correspond to three species of Nkx-1.2 mRNA that are formed by alternative splicing at conventional 5′ donor and 3′ acceptor splice sites; however, seven cDNA clones were found that correspond to three species of Nkx-1.2 mRNA from testes that have novel TG/AC 5′ and 3′ splice sites. The consensus splice sequences are: 5′ donor, CC↓TGGAAG; 3′ acceptor, ACTTAC↓. Predicted amino acid sequences suggest that some transcripts may be translated into proteins that lack part or all of the homeodomain. At least three bands of Nkx-1.2 mRNA were found in RNA from the testes. Nkx-1.2 mRNA was shown to be present in postmeiotic germ cells of the testis and in mature spermatozoa. Nkx-1.2 mRNA also was found in regions of the adult cerebral cortex, hippocampus, diencephalon, pons/medulla, and cerebellum. Nkx-1.2 mRNA was found in embryos in highest abundance in 10-day embryos; the mRNA levels decrease during further development. Nkx-1.2 mRNA also was found in discrete zones of the embryonic mesencephalon and myelencephalon.