Transcript localizations for Mox genes have implicated this homeobox gene subfamily in the early steps of mesoderm formation. We have extended these studies by determining the protein expression profile of Mox-1 and Mox-2 during mouse development. The time of onset of Mox protein expression has been accurately obtained to provide clues as to their roles during gastrulation. Expression of Mox-1 protein is first detected in the newly formed mesoderm of primitive streak stage mouse embryos (7.5 days post-coitum, d.p.c.). In contrast, Mox-2 protein is first detected at 9.0 d.p.c. in thr already formed somites. Additionally, immunostaining reveals new and distinct areas of Mox expression in the branchial arches and limbs that were not reported in our previous mRNA localization analysis. Mouse Mox-2 antibodies cross-react specifically in similar embryonic tissues in chick indicating the conservation of function of Mox genes in vertebrates. These expression data suggest that the Mox genes function transiently in the formation of mesodermal and mesenchymal derivatives, after their initial specification, but before their overt differentiation. Furthermore, while there appears to be some overlap in protein expression between Mox-1 and Mox-2 during somitogenesis, unique areas of expression indicate several distinct roles for the Mox genes during development.