In the presence of retinoic acid (RA), F9 murine teratocarcinoma cells differentiate into cells resembling the extra-embryonic endoderm of the early mouse embryo. Using differential hybridization, we have cloned and characterized six cDNAs corresponding to mRNAs that exhibit reduced expression in F9 cells following RA treatment. Two of these cDNAs encode novel genes (REX-2 and REX-3). The other isolated cDNAs encode genes that have been previously described in other contexts: 1-4 (cyclin D3); 2-10 (pyruvate kinase); 2-12 (glutathione S-transferase); and 2-17 (GLUT 3). The mRNA levels of these genes are reduced by RA or RA plus theophylline and cAMP (RACT) only after 48 h of treatment, and continue to decrease at 96 h. The half-lives of these mRNAs are not changed by RA treatment, indicating that these mRNAs may be regulated through a transcriptional mechanism. In isoleucine-deprived cells, which are growth arrested but do not differentiate, the steady state mRNA levels of genes Rex 2, Rex 3, pyruvate kinase and GLUT 3 are not reduced, in contrast to cyclin D3 and glutathione S-transferase. The expression of the REX-2, REX-3, pyruvate kinase, glutathione S-transferase and GLUT 3 genes is reduced by RACT to the same extent in F9 RARgamma-/- and RARalpha-/- lines as in F9-Wt. In contrast, cyclin D3 exhibits lower mRNA expression in F9 RARgamma-/- and RARalpha-/- stem cells, and this mRNA is not decreased by RACT treatment. Overexpression of cyclin D3 blocks the RA-induced growth arrest of F9 cells, indicating that the downregulation of this gene following RA treatment may constitute a necessary step in the cascade of events leading to growth inhibition by RA.