BACKGROUND: The low plasma triiodothyronine (T3) observed during prolonged critical illness can be explained in part by suppressed hepatic deiodinase type I (D1) and increased D3 activity. Infusion of thyrotropin-releasing hormone (TRH) can restore D1 and D3 activity in critically ill rabbits, but it remains unknown whether this is a direct effect of TRH or the TRH-induced rise in circulating thyroxine (T4) and T3. METHODS: To answer this specific question, burn-injured rabbits randomly received a 4-day treatment with saline, T4, T3, T4+T3, or TRH, started on day 4 of the illness. Plasma iodothyronine concentrations, D1 and D3 activity, and T3-responsive gene expression were quantified in liver and kidney. RESULTS: Infusion of T4, T3, or TRH increased circulating T3 levels and hepatic D1 activity. Co-infusion of T3 with T4 enhanced T4 to T3 conversion as demonstrated by lower T4, higher T3, and lower reverse T3 (rT3) levels and tended to further increase hepatic D1 activity. Hepatic D1 activity correlated positively with circulating T3 and the T3/rT3 ratio, but not with T4, rT3, or thyroid-stimulating hormone. CONCLUSIONS: During prolonged critical illness, D1 activity is primarily regulated via changes in circulating T3, suggesting that the low plasma T3 concentrations may be important in sustaining low D1 activity in this condition.