BACKGROUND: Lithium (Li) has been associated with cardiac teratogenicity in the developing fetus. We took advantage of the association of therapeutic administration of Li with an increase in heart defects to gain insight into both normal and pathological heart and valve development with GSK-3 inhibition. The objective of this study was to define whether Li mimicry of canonical Wnt/β-catenin signaling induces cardiac valve defects. METHODS: Li was administered by a single intraperitoneal injection to the pregnant mouse on embryonic day E6.75, much earlier than heretofore analyzed. On E15.5 developing heart defects were defined by Doppler ultrasound. The embryonic hearts were analyzed for changes in patterning of active canonical Wnt expression and nuclear factor of the activated T cells-c1 (NFATc1), both key regulators of valve development. Li-exposed chick embryos were used to define the early cell populations during gastrulation that are susceptible to GSK-3 inhibition and may relate to valve formation. RESULTS: Li exposure during gastrulation decreased the number of prechordal plate (PP) cells that reached the anterior intestinal portal, a region associated with valve development. Li decreased expression of Hex, an endoderm cardiac inducing molecule, normally also expressed by the PP cells, and of Sox 4 at the anterior intestinal portal and NFAT, critical factors in valvulogenesis. CONCLUSIONS: Cells existing already during gastrulation are associated with valve formation days later. The Wnt/β-catenin signaling in PP cells is normally repressed by Wnt antagonists and Hex is up-regulated. The antagonism occurring at the receptor level is bypassed by Li exposure by its intracellular inactivation of GSK-3 directly to augment Wnt signaling. Birth Defects Research (Part A), 2008. © 2008 Wiley-Liss, Inc.