A putative mouse homeobox gene (Hox-3.4) was previously identified 4kb downstream of the Hox-3.3 (Hox-6.1)* gene (Sharpe et al. 1988). We have now sequenced the Hox-3.4 homeobox region. The predicted amino acid sequence shows highest degree of homology in the mouse with Hox-1.3 and -2.1. This, together with similarities in the genomic organisation around these three genes, suggests that they are comembers of a subfamily, derived from a common ancestor. Hox-3.4 appears to be a homologue of the Xenopus Xlhbox5 and human cp11 genes (Fritz and De Robertis, 1988; Simeone et al. 1988). Using a panel of mouse-hamster somatic cell hybrids we have mapped the Hox-3.4 gene to chromosome 15. From the results of in situ hybridization experiments, we describe the distribution of Hox-3.4 transcripts within the 12 1/2 day mouse embryo, and we compare this with the distributions of transcripts shown by seven other members of the Hox gene network. We note three consistencies that underlie the patterns of expression shown by Hox-3.4. First, the anterior limits of Hox-3.4 transcripts in the embryo are related to the position of the Hox-3.4 gene within the Hox-3 locus. Second, the anterior limits of Hox-3.4 expression within the central nervous system are similar to those shown by subfamily homologues Hox-2.1 and Hox-1.3, although the tissue-specific patterns of expression for these three genes show many differences. Third, the patterns of Hox-3.4 expression within the spinal cord and the testis are very similar to those shown by a neighbouring Hox-3 gene (Hox-3.3), but they are quite different from those shown by Hox-1 genes (Hox-1.2, -1.3 and -1.4).