We have studied the rat malic enzyme gene as a model for thyroid hormone (triiodothyronine (T3)) regulation of transcription. Our previous studies showed that transcription of this gene is controlled by T3 in vivo. In this study, we used COS-7 cells in culture to determine the location of a T3 response element (TRE) within this gene. Cotransfection of the rat T3 alpha-receptor cDNA with a reporter gene linked to 5'-deletion mutants of the malic enzyme gene 5'-flanking region revealed the presence of a TRE between positions -315 and -248. Using T3 receptors synthesized in vitro from their cDNAs, we have identified, through the use of gel shift assays and footprinting, a single DNA-binding site (positions -281 to -261) for the receptor within the rat malic enzyme gene TRE. The site was capable of binding either the rat alpha- or human beta-receptor with similar affinities. Competition binding studies indicated that the apparent affinity of receptor binding to the malic enzyme gene was similar to that of the rat alpha-myosin heavy chain gene TRE (positions -151 to -122) and was significantly greater than that of the rat growth hormone TRE (positions -192 to -163). These results indicate that both the alpha- and beta-forms of the nuclear T3 receptor are capable of binding directly to the malic enzyme gene 5'-flanking region at a site which functions as a hormone-inducible cis-regulatory element. In conjunction with our previous finding that T3 induces or activates essential transcription factors which bind to the promoter, it appears that the regulation of transcription of the malic enzyme gene by thyroid hormone involves at least two regulatory pathways.