Studies were conducted to determine if the expression of the gene for retinol-binding protein (RBP) and/or transthyretin (TTR) could be induced upon differentiation of F9 teratocarcinoma cells to either visceral endoderm or parietal endoderm. Both TTR mRNA and RBP mRNA were undetectable in the undifferentiated F9 stem cells and in F9 cells differentiated to parietal endoderm. However, TTR mRNA and RBP mRNA were both detected in F9 cell aggregates differentiated to embryoid bodies (which contain visceral endoderm-like cells) by treatment of the aggregates in suspension with retinoic acid. TTR mRNA was observed at 3 days, and RBP mRNA at 5 days, after treatment of the F9 cell aggregates with retinoic acid. Both TTR mRNA and RBP mRNA were found to be specifically localized by in situ hybridization in the outer layer of cells (the visceral endoderm-like cells) of the embryoid bodies. Finally, synthesis and secretion of both RBP and TTR by F9 cell embryoid bodies was demonstrated by specific immunoprecipitation of each newly synthesized protein from the culture medium. These data thus demonstrate the production and presence of RBP mRNA and TTR mRNA, and the synthesis and secretion of RBP and TTR, by F9 cell embryoid bodies (specifically by visceral endoderm-like cells). This finding suggests that these two proteins may be synthesized by rodent embryos extremely early in embryonic development.