There is limited in vivo data in humans evaluating myocardial fat utilization during increased heart work. This study was done to determine myocardial free fatty acid FFA metabolism during rest and atrial pacing, which does not change arterial substrate concentration. We studied 7 healthy men and women (age=49.7±3.9 yr; BMI=23.4±1.1 kg/m², VO₂max=35.5±3.0 ml/kg/min, ejection fraction=68±3%). After 3 days of dietary control, coronary sinus, femoral arterial and venous, and peripheral venous catheters were placed. Subjects received ¹³C-bicarbonate, followed by a continuous infusion of [1-¹³C] palmitate through the end of the study. Arterial and coronary sinus blood sampling and measurements of resting coronary sinus blood flow were made during rest and atrial pacing to 120 bpm. Myocardial MVO₂ increased (p<0.05) from rest to atrial pacing. Coronary sinus FFA concentration was significantly lower than arterial through rest and atrial pacing (p=0.007). Isotopically measured myocardial palmitate uptake increased significantly from rest to atrial pacing (p=0.031). Approximately one third of palmitate delivery was extracted by the myocardium during rest and atrial pacing. Myocardial V¹³CO₂ production and palmitate oxidation increased significantly from rest (p<0.01) to atrial pacing. Net glycerol balance was significantly greater than zero during rest (p=0.04), but not different than zero during atrial pacing (p=0.13). These data suggest myocardial lipid uptake and oxidation increase with greater heart work during atrial pacing, with a similar relative proportion of fat oxidation to total myocardial energy expenditure.