Hoxa-9 is a homeobox-containing gene located in the HoxA cluster on chromosome 6 in mice. To identify the temporal and spatial expression patterns of Hoxa-9 during mouse development, we subjected it to Northern blot and in situ hybridization. Since we have previously shown that human Hoxa-9 contains 3 exons which are differentially expressed in different fetal and adult tissues (Min et al., 1998), three different exon-specific probes were prepared and used in the analysis. Northern and in situ data showed that exon I was not used in mice, whereas exons II and III gave the same Northern and in situ data., meaning that both were used to produce the same transcript. A 1.8kb transcript and two minor (3.2 and 7.0kb) transcripts were produced at day 7.0 post coitum (p.c.) through until day 12.0 p..c., and then disappeared by day 15.0 p.c. In situ analysis demonstrated that the expression started during the early onset of gastrulation around day 7.5 p.c. with a uniform expression pattern along the embryonic and extraembryonic tissues. The expression pattern eventually became restricted to a small subset of tissues, such as limb and axial skeleton by day 14.5 p.c. The Hoxa-9 gene was expressed in mesoderm and ectoderm derivatives and particularly strong expression was detected in the brain, the posterior dorsal part of the spinal cord, spinal ganglia, heart, liver, kidney, lung, digestive system and limb buds during the mid-gastrulation stage. Our data suggested that the temporal and spatial regulation of Hox genes is not limited to the axial CNS and skeleton, In the trunk, Hoxa-9 was expressed according to a pattern without any correlation to the anterior-posterior axis. Differential expression was also observed along the dorso-ventral and proximo-distal axis, which implies that spatial regulation of gene expression follows different positional cues on different axes.