We have studied the effect of zinc removal on thyroid hormone (T3) induction of growth hormone (GH) gene expression in rat pituitary tumor (GH3) cells. It was hypothesized that zinc chelation disrupts thyroid hormone receptor (TR) function and thus blocks T3 induction of GH gene expression. However, previous studies in our laboratory have shown that chelation of zinc with diethylenetriaminepentaacetic acid (DTPA) amplifies T3 induction of GH mRNA. We have sought to explain these findings and further probe the interactions of T3 and zinc. GH3 cells treated with a second chelator EDTA show a dose dependent induction of GH3 mRNA. However, EDTA is less potent than DTPA. DTPA also amplifies the effect of 9-cis retinoic acid (9-cis RA) on GH mRNA induction suggesting it can operate through other ligands in the T3 signaling pathway. Time course and zinc reversibility studies show a lag time of 24 hrs to see the DTPA response and to reverse the effect with zinc. A zinc sensitive accessory protein or the downregulation of a repressor protein may be necessary to enhance transcription. GH3 cells treated with cycloheximide, a translational inhibitor, blunted the T 3 and DTPA response in GH mRNA, suggesting that protein synthesis is required. Studies with the transcriptional inhibitor actinomycin D demonstrated that GH mRNA was less stable in the presence of DTPA than its absence, eliminating mRNA stabilization as a mechanism for the DTPA effect. Transient transfection assays with the GH promoter ligated to a luciferase reporter gene showed that DTPA decreased luciferase expression compared to T3 treated cells. Concurrent addition of zinc restored luciferase expression. Human TR were expressed in bacteria and purified with Ni-affinity chromatography. Hormone binding studies indicated the purified TR was a physiologically relevant form of the protein. This protein is now available to investigate direct zinc effects on T3/TR/DNA binding and further characterize the role of zinc in the T3 signaling pathway.