The human DIO3 gene and its mouse homolog, Dio3, map to chromosomes 14q32 and 12F1, respectively, and code for the type 3 deiodinase, an enzyme that inactivates thyroid hormones and is highly expressed during pregnancy and development. Mouse Dio3 is imprinted and preferentially expressed from the paternal allele in the fetus. We analyzed the human DIO3 genomic region and identified a gene (DIO3OS) that is transcribed in the antisense orientation. Multiple DIO3OS transcripts are expressed in most tissues. The structure of several DIO3OS cDNAs obtained by RT-PCR-based techniques reveals the occurrence of numerous splice variants. The exon–intron structures of DIO3OS are similar in mouse and human, but the homology of the exonic sequence is very low, except for the first exon, and no conserved open reading frame is present. We also detected DIO3 transcripts containing additional 5′ untranslated sequence and a potential alternative upstream promoter for mouse Dio3. Exonic sequence of a Dio3os cDNA overlaps with the Dio3 promoter and strong promoter activity in the antisense orientation is detected in a genomic fragment located 3′ of mouse and human DIO3 but not in the DIO3 promoter region. These results suggest that the DIO3 gene may lie within the structure of the antisense gene, a complex arrangement often observed in imprinted loci.