Objective: To investigate whether triiodothyronine pretreatment enhanced beta-adrenergic responses in stunned myocardium and whether this acute effect of triiodothyronine was mediated through the cyclic adenosine 3′,5′-monophosphate (AMP) system. Design: A prospective study. Setting: University laboratory. Participants: Rabbits. Interventions: Rabbit ventricular myocytes were isolated and placed in a medium equilibrated with either air (control) or with 95% N 2 and 5% CO 2 (stunned) for 15 minutes at 37°C. The stunned myocytes were reoxygenated with air for 30 minutes. Triiodothyronine (10 nmol/L) and/or isoproterenol (0.1 nmol/L) was added to the myocytes. Myocyte shortening was measured by using a video-edge detector. Measurements and Main Results: In electrically stimulated cells, the basal values of the percent shortening (22%-30%) and the maximum rate of shortening (22%-25%) were significantly reduced in the stunned myocytes. Isoproterenol (5 minutes) alone significantly increased the percent shortening in the control (from 3.70 ± 0.36 to 4.14 ± 0.37) but not in the stunned myocytes (from 2.60 ± 0.30 to 3.15 ± 0.27). Triiodothyronine (5 minutes) alone significantly increased the percent shortening in the control (from 3.75 ± 0.36 to 4.34 ± 0.45) and in the stunned myocytes (from 2.91 ± 0.2 to 3.85 ± 0.26). After triiodothyronine pretreatment for 5 minutes, isoproterenol caused greater increases in the percent shortening in both the control (37%) and the stunned myocytes (62%) than either agent alone. Isoproterenol or triiodothyronine caused small increases in the maximum rate of shortening in the control (14%-16%) and the stunned myocytes (34%-49%). After triiodothyronine pretreatment, isoproterenol caused greater increases in the maximum rate of shortening in both groups (control: 41%, stunned: 73%) than either agent alone. Isoproterenol caused an increase in the level of cyclic AMP (ρ;moles/10 5 myocytes) in the control (from 2.92 ± 0.47 to 3.77 ± 0.43) but not in the stunned myocytes (from 2.42 ± 0.25 to 2.42 ± 0.20). Triiodothyronine pretreatment did not cause any change in cyclic AMP levels in the control (2.50 ± 0.29) or in the stunned myocytes (2.60 ± 0.40). After triiodothyronine pretreatment, isoproterenol caused a small increase in the cyclic AMP level in the control but not in the stunned myocytes. Conclusions: The data showed that the myocardial beta-adrenergic responses were more sensitive to ischemic insult than the triiodothyronine responses. Triiodothyronine pretreatment enhanced beta-adrenergic responses in both the control and the stunned myocytes. However, this acute positive inotropic effect of triiodothyronine might not be mediated through the cyclic AMP system.