The S8 gene of the mouse contains a paired-like homeobox and is expressed during organogenesis in a complex pattern in mesenchyme. Here we show that S8 is first expressed in extraembryonic mesoderm of primitive streak stage embryos and that it is strongly expressed in mesoderm of early-headfold/neural plate stage embryos. It is, however, absent from the primitive streak and its immediately adjacent areas. Early after initial mesoderm formation, this expression domain defines a specific region of the mesoderm that, to some extent, foreshadows the later S8 expression pattern. The pattern at this stage is roughly complementary to that of the brachyury (T) gene. Expression in cell lines was studied both at the level of RNA and protein. Mesoderm specificity of S8 expression was confirmed in terminally differentiated cell lines. S8 is activated by retinoic acid in certain embryonal carcinoma cells, embryonic stem cells, and neural crest cell lines, and also during spontaneous differentiation in embryonic stem cells.