Retinoic acid (RA)-induced endodermal differentiation of F9 teratocarcinoma cells is accompanied by altered of many genes, including the retinoic acid receptor (RAR) alpha, beta, and gamma and retinoic x receptor (RXR) alpha and gamma genes. In addition, RA enhances the binding of nuclear receptors to the RA responsive elements and increases the trans-activation of the RA-responsive genes in F9 cells. These data suggest that RA increases the overall function of RA receptors in F9 cells. However, the cell line F9E, established by long-term exposure of F9 cells to RA, is insensitive to RA. These cells expressed the genes for keratin 8 and tissue specific plasminogen activator (t-PA), which are characteristic of endoderm cells. Northern blot analysis showed that F9E cells expressed high basal levels of RAR and RXR mRNAs, but genes including the RARs and RXRs, which are normally regulated by RA in F9 cells, were no longer regulated in F9E cells. Further, F9E cells were not sensitive to the antiproliferative effect of RA. In these cells, high levels of RA receptors constitutively bound to RA responsive elements, but RA could neither increase the amount of RA receptor binding to the RA responsive elements, nor enhance the transcription of the RA target genes. These findings provide a possible explanation for insensitivity of endodermal cells to the actions of RA. Our data also indicate that the levels of endogenous RA receptors do not predict the degree of RA sensitivity, and that RA responsiveness might be a function of the cell's ability to regulate RA receptor genes.