There is minimal in vivo data in humans evaluating myocardial substrate utilization during increased heart work. This study was performed to determine the balance of myocardial glucose and lactate metabolism during rest and increased heart work induced by atrial pacing in seven healthy men and women (age, 49.7 +/- 3.9 years; body mass index, 23.4 +/- 1.1 kg m(-2), maximum oxygen consumption, 35.5 +/- 3.0 ml kg(-1) min(-1), ejection fraction, 68 +/- 3%). After 3 days of dietary control, catheters were placed in coronary sinus, femoral arterial and venous, and peripheral venous blood vessels. Subjects received a primed continuous infusion of [3,3,3-(2)H]lactate and [6,6-(2)H]glucose throughout the study. Arterial and coronary sinus blood sampling and measurements of coronary sinus blood flow were made during rest and atrial pacing at approximately 111 beats min(-1). Myocardial oxygen consumption increased (P = 0.04) from rest to atrial pacing. Net glucose uptake increased (P = 0.04) from rest to atrial pacing with unchanged fractional extraction (rest: 9.1 +/- 2.7%, atrial pacing 9.8 +/- 2.9%). The percentage of whole body glucose disposal from myocardial uptake also increased from rest to atrial pacing. Isotopically measured lactate uptake also increased significantly from rest to atrial pacing with no significant differences in fractional extraction. The myocardium released lactate throughout the experiment, which increased significantly from rest and atrial pacing (P < 0.05). The heart accounted for a significantly greater percentage of whole body lactate disposal during atrial pacing (15.0 +/- 4.4%) compared to rest (4.9 +/- 0.9%, P = 0.03). These data suggest: (1) in the absence of ischaemia the myocardium is constantly taking up and releasing lactate at rest which increases during atrial pacing, and (2) when arterial substrate delivery is unchanged, increased myocardial work is accomplished with similar proportions of glucose and lactate utilization in healthy humans in vivo.