Disruption of thyroid hormone (TH) signaling can compromise vital processes both during development and in the adult. We previously reported on high-throughput screening experiments for man-made TH disruptors using a stably integrated line of rat pituitary cells, GH3.TRE-Luc, in which a thyroid hormone receptor (TR) response element drives luciferase (Luc) expression. In these experiments, several retinoid/rexinoid compounds activated the reporter. Here we show that all-trans and 13-cis retinoic acid appear to function through the heterodimer partners of TRs, retinoid-X receptors (RXRs), as RXR antagonists abrogated retinoid-induced activation. The retinoids also induced known endogenous TR target genes, showing good correlation with Luc activity. Synthetic RXR-specific agonists significantly activated all tested TR target genes, but interestingly, retinoid/rexinoid activation was more consistent between genes than the extent of T3-induced activation. In contrast, the retinoids neither activated the Luc reporter construct in transient transfection assays in the human hepatocarcinoma cell line HuH7, nor two of the same T3-induced genes examined in pituitary cells. These data demonstrate the suitability and sensitivity of GH3.TRE-Luc cells for screening chemical compound libraries for TH disruption and suggest that the extent of disruption can vary on a cell type and gene-specific bases, including an underappreciated contribution by RXRs.