A novel family of genes, characterized by the presence of a region of homology to the DNA-binding domain of the Brachyury (T) locus product, has recently been identified. The region of homology has been named the T-box, and the new mouse genes that contain the T-box domain have been named T-box 1-6 (Tbx1 through Tbx6). As the basis for further study of the function and evolution of these genes, we have examined the expression of 5 of these genes, Tbx1-Tbx5, across a wide range of embryonic stages from blastocyst through gastrulation and early organogenesis by in situ hybridization of wholemounts and tissue sections. Tbx3 is expressed earliest, in the inner cell mass of the blastocyst. Four of the genes are expressed in different components of the mesoderm or mesoderm/endoderm during gastrulation (Tbx1 and Tbx3-5). All of these genes have highly specific patterns of expression during later embryogenesis, notably in areas undergoing inductive tissue interactions. In several cases there is complementary expression of different genes in 2 interacting tissues, as in the lung epithelium (Tbx1) and lung mesenchyme (Tbx2-5), and in mammary buds (Tbx3) and mammary stroma (Tbx2). Tbx1 shows very little overlap in the sites of expression with the other 4 genes, in contrast to a striking similarity in expression between members of the 2 cognate gene sets, Tbx2/Tbx3 and Tbx4/Tbx5. This is a clear reflection of the evolutionary relationship between the 5 genes since the divergence of Tbx1 occurred long before the relatively recent divergence of Tbx2 and 3 and Tbx4 and 5 from common ancestral genes. These studies are a good indication that the T-box family of genes has important roles in inductive interactions in many stages of mammalian embryogenesis.