Hoxb3 plays important roles in embryogenesis and it has a complex transcription profile of mRNAs, non-coding RNAs and anti-sense RNAs. Characterization of the spatial expression patterns of these RNAs is important to understand their functions. We investigated the regulation and spatial expression patterns of multiple RNA transcripts derived from the Hoxb3 gene locus. By 5'-RACE we identified four novel transcription initiation sites and initiating exons, and by luciferase activity assay we identified a new promoter region. Expression pattern analysis of the alternative transcripts containing specific initiation exons in mouse embryos suggests that there are co-operations between the initiation exons, their adjacent promoters and enhancer elements to orchestrate overlapping neural tube specific transcription profiles for Hoxb3. Furthermore, we showed that anti-sense transcripts derived from the Hoxb3 locus were expressed in the hindbrain with distinct rhombomere boundaries, in a pattern complementary to the sense coding mRNA transcripts. Our results suggest that the multiple non-coding RNAs could be involved in the regulation of Hoxb3. (C) 2010 Elsevier Inc. All rights reserved.