Retinoic acid (RA) induces F9 cells, the mouse teratocarcinoma cells, to differentiate into primitive endoderm and further into visceral and parietal endoderm depending on the culture conditions. To elucidate the instructive mechanisms involved in the differentiation steps we investigated the effects of Wnt-signaling members, Wnt3a and beta-catenin, on the differentiation of F9 cells and beta-catenin-deficient F9 cells (beta T cells). RA up-regulated the expression of differentiation markers for primitive, visceral and parietal endoderm in F9 cells but not for visceral endoderm in beta T cells. Wnt3a or leukemia inhibitory factor (LIF) inhibited the RA-induced differentiation in F9 cells. LIF but not Wnt3a Could inhibit differentiation in beta T cells. RA evoked ZO-1 alpha+ signals at cell-to-cell contacts in F9 cells in a Wnt3a sensitive manner. The results suggest that Wnt3a inhibits differentiation into endoderm through a pathway involving beta-catenin, and beta-catenin might be necessary in the process leading from primitive to visceral endoderm in F9 cells. (C) 2009 Elsevier Inc. All rights reserved.