Classical embryology experiments have indicated the existence of dorsal-type and ventral-type mesoderms that arise as a consequence of mesoderm induction during vertebrate development. Here we report that the zebrafish tbx6 gene, a member of the Brachyury-related T-box family of genes, is exclusively expressed by ventral mesendoderm. Three observations link the expression of tbx6 to ventral mesoderm specification. First, the gene is initially expressed at the onset of gastrulation within a ventrolateral subpopulation of cells that express the pan-mesodermal gene, no tail (Brachyury). Second, the mesoderm-inducing factors activin and bFGF activate tbx6 expression in animal caps. Third, dorsalization of the mesendodermal precursor population following exposure of embryos to lithium ions causes down-regulation of tbx6 transcription. tbx6 is expressed transiently in the involuting derivatives of the ventral mesendoderm, which give rise to nonaxial mesodermal tissues; its expression is extinguished as tissue differentiation progresses. Transcription of tbx6 commences about an hour after initiation of expression of the pan-mesendodermal gene no tail and the organizer gene goosecoid. The dependence of tbx6 expression on no tail activity was examined in no tail mutant embryos. The activation of tbx6 transcription in ventral mesoderm does not depend on no tail gene activity. However, no tail appears to contribute to the maintenance of normal levels of tbx6 transcription and may be required for tbx6 transcription in the developing tail.