The mechanism of ST-segment depression during exercise electrocardiographic treadmill testing (ETT) is unknown. The relatively good correlation between the results of ETT and myocardial perfusion imaging suggests that ST-segment depression may be related to the magnitude of ischemia. Previous studies that investigated this relation have produced conflicting results. We evaluated 1,006 patients who underwent symptom-limited ETT and technetium-99m single-photon emission computed tomographic myocardial perfusion imaging at rest and during stress at a single institution. The magnitude, extent, and duration of ST-segment depression were each strongly associated with the magnitude of myocardial ischemia (p <0.001 for all). The magnitude, extent, and duration of ST-segment depression were highly correlated with each other and had similar relations to the magnitude of ischemia. After adjustment for significant clinical and ETT parameters, these relations remained highly significant. The location of myocardial ischemia was unrelated to ST-segment depression. This large study found that ST-segment depression during ETT is strongly associated with the magnitude of ischemia. These data support a causative role for the magnitude of ischemia in the generation of ST-segment depression.