Malformation of the cardiac valves and septa accounts for a large proportion of congenital heart defects. Mutations in the human cysteine-rich with EGF-like domains 1 (CRELD1) gene have been described as a risk factor for the pathogenesis of atrioventricular septum defects (AVSD) (1). However, the physiological function of CRELD1 is not known. We generated Creld1 knockout-mice (Creld1KO) to unravel the role of Creld1 in heart development. We reveal that Creld1 is an essential regulator of the calcineurin/NFATc1 signaling pathway (2), which is crucial for cardiac development. Calcineurin is a phosphatase consisting of a catalytic A and a regulatory B subunit (3), which dephosphorylates the transcription factor NFATc1 in the cytoplasm, thereby promoting its translocation to the nucleus (4).Our results demonstrate that Creld1 interacts with the regulatory calcineurin B subunit, thereby, controlling the phosphatase activity of the catalytic A subunit. We analyzed the effect of Creld1 on calcineurin function using the translocation of NFATc1 to the nucleus as a read-out (Figure 1). When expressed in cells, NFATc1 mainly remained in the cytoplasm, only 6 % of cells displayed NFATc1 localization in the nucleus. Stimulation with thapsigargin (Tg) activates endogenous calcineruin and caused NFATc1 translocation to the nucleus (75 %). Similarly to Tg, the presence of Creld1 dramatically increased the number of cells that showed nuclear NFATc1 localization (51 %). Treatment of Creld1-expressing cells with cyclosporine A (CsA), a potent calcineurin inhibitor, strongly reduced the number of cells showing NFATc1 in the nucleus (1 %). The function of Creld1 is particularly important in the cells of the endocardial cushion, the precursor of the heart valve: whereas in wild-type embryos at developmental day E10.5, NFATc1 translocates to the nucleus, in Creld1KO embryos NFATc1 remains in the cytoplasm. Thereby, the expression of NFATc1 target-genes is diminished and cell proliferation in the endocardial cushion is abolished. As a result, cushion formation is impaired and Creld1KO embryos die at E11.0. To analyze whether the two missense-mutations Creld1R107H and Creld1R329C in the human CRELD1 gene associated with AVSD (1,5) affect the action of Creld1 on calcineurin/NFATc1 signaling, we introduced the corresponding mutations into the murine Creld1 gene. Both point mutations dramatically reduced the translocation of NFATc1 to the nucleus and, thereby, NFAT-dependent gene expression (Figure 1). In summary, our results demonstrate that Creld1 controls the calcineurin-dependent translocation of NFATc1 to the nucleus and that mutations in Creld1 interfere with the action of Creld1 on calcineurin function. Thus, our results provide first insights into how mutations in CRELD1 affect cellular signaling and, thereby, the formation of cardiac valves.