A 6-kb antisense transcript (Dnm3os) contained within an intron of the mouse Dnm3 gene has been identified in a screen for genes that may be regulated by the basic helix-loop-helix transcription factor Twist during mouse development. The antisense transcript is highly conserved between vertebrate species, but does not appear to encode a protein. We show that expression of the Dnm3 and Dnm3os transcripts overlaps during embryogenesis and in adult tissues, except that Dnm3 is most highly expressed in adult brain and testis and expressed at lower levels in embryos, whereas the antisense transcript is most strongly expressed in embryos and gravid uterus. Both Dnm3 and Dnm3os are downregulated in branchial arch tissue of Twist-null embryos. The conservation and restricted expression pattern of this noncoding transcript suggest that it may perform an important function during embryonic development and further suggest a connection between the regulation of Dnm3 and this novel intronic, antisense transcript.