In this study, we examined whether gene trap methodology, which would be available for systematic identification and functional analysis of genes, is effective for screening of Wnt-responsive genes during mouse development. We screened out two individual clones among 794 gene-trapped embryonic stem cell lines by their in vitro response to WNT-3A proteins. One gene was mainly expressed in the ductal epithelium of several developing organs, including the kidney and the salivary glands, and the other gene was expressed in neural crest cells and the telencephalic flexure. The spatial and temporal expression of these two genes coincided well with that of several Wnt genes. Furthermore, the expression of these two genes was significantly decreased in embryos deficient for Wnts or in cultures of embryonic tissues treated with a Wnt signal inhibitor. These results indicate that the gene trap is an effective method for systematic identification of Wnt-responsive genes during embryogenesis. Developmental Dynamics 233:484–495, 2005. © 2005 Wiley-Liss, Inc.