Gnot1, a Not (for notochord) family homeobox gene, is expressed in the chick pregastrulation blastoderm. Gnot1 expression in the epiblast is upregulated as the posteriorly derived hypoblast moves forward anteriorly to form a layer beneath it, which is of particular interest considering the known inductive role of the ast in axis formation in the chick. Both activin and retinoic acid are able to activate Gnot1 expression in cultured blastodermal cells and show a strong synergistic effect when applied in combination. Strong superinduction of Gnot1 transcripts in the presence of cycloheximide also indicates the presence of a potent and labile intracellular inhibitor capable of modulating Gnot1 expression. During gastrulation, Gnot1 transcripts become localized specifically to tissues associated with 'organizer' function (Hensen's node, head process, notochord). The expression data and the response to mesoderm inducing factors and axial 'caudalizing' signals suggest that Gnot1 may be involved in specification of the embryonic body axis and could play a part in regulating features of the trunk/tail organizer in the chick embryo. © 1995 by Academic Press, Inc.