In the heart elevated thyroid hormone leads to both an upregulation of metabolic pathways associated with energy production and the development of hypertrophy. The malate/aspartate shuttle, necessary to transfer cytosolic reducing equivalents into the cardiac mitochondria, is increased 33% in hyperthyroid rats. Of the shuttle's two inner mitochondrial membrane protein transporters, the aspartate-glutamate carrier is rate limiting. The excitatory amino acid transporter type 1 (EAAT1) has recently been shown to function as a glutamate carrier in the malate/aspartate shuttle. We hypothesize that EAAT1 is regulated by thyroid hormone. Methods: Adult Sprague-Dawley rats were injected with triiodothyronine (T3) or saline over a period of 8-9 days or provided propylthiouracil (PTU) in their drinking water for two months. Serum free T3 levels were measured. Rats were euthanized, hearts weighed, and tissue either frozen or processed for mitochondria studies. Steady-state mRNA levels of EAAT1 and the other cardiac mitochondrial aspartate-glutamate transporters aralar1 and citrin were determined by Northern blot analysis and normalized to 18S rRNA. A spectrophotometric assay of maximal malate/aspartate shuttle activity was performed on isolated cardiac mitochondria from PTU-treated and control animals. Protein lysates from mitochondria were separated by SDS-PAGE and probed with a human anti-EAAT1 IgG. Results: Compared to control myocardium, EAAT1 steady-state mRNA levels were increased nearly three-fold in the T3-treated rats (3.1±0.5 vs. 1.1±0.2; P<0.05) and decreased in PTU-treated myocardium (2.0±0. 3 vs. 5.2±1; P<0.05). mRNA levels of aralar1 were unchanged in the T3 treated animals and somewhat decreased in the PTU-treated animals (7.1±1.0 vs. 9.3±0.1, P<0.05). Citrin mRNA levels were decreased in T3 treated animals, and unchanged in PTU-treated animals. EAAT1 protein level was increased in cardiac mitochondria isolated from T3-treated rats compared to controls (8.9±0.4 vs. 5.9±0.6; P<0.005) and unchanged in PTU-treated mitochondria. No difference in malate/aspartate shuttle capacity was found in cardiac mitochondria between the PTU-treated and control rats. Conclusions: Hyperthyroidism in rats is related to an increase in cardiac expression of both EAAT1 mRNA and protein. The 49% increase in EAAT1 mitochondrial protein level correlates with the previously published increase in malate/aspartate shuttle activity in cardiac mitochondria from hyperthyroid rats. While hypothyroidism resulted in a decrease in EAAT1 mRNA, neither the EAAT1 protein level nor shuttle activity was affected. The regulation of EAAT1 by thyroid hormone may facilitate increased metabolic demands of the cardiomyocyte during hyperthyroidism and impact cardiac function in the setting of hyperthyroidism.