One of the prominent markers of heart failure at the molecular level is a decrease in function and/or expression of the sarcoplasmic reticulum ATPase protein (SERCA). It has been often postulated that a decrease in SERCA pump activity can contribute in a major way to decreased cardiac function. To establish a functional relationship, we assessed how alterations in SERCA activity level affect basic contractile function, in healthy myocardium devoid of other significant molecular changes. We investigated baseline contractile function, frequency-dependent activation, and -adrenergic response in ultra-thin trabeculae isolated from hearts of mice expressing SERCA1a (TG), under-expressing SERCA2a (HET), and their respective wild-type littermates (WT). At physiological temperature and frequency, compared to their respective WT, SERCA1a mice displayed increased developed force at frequencies 4-8 Hz (~90% increase at 4 Hz), and equal force to WT at 10-14 Hz. Force development at 4 Hz in presence of other 1 µM isoproterenol was similar in TG and WT mice. In HET mice, developed force was nearly identical at lower end of frequency range (4-8 Hz), but slightly depressed at higher range (P<0.05 at 14 Hz). In presence of 1 µM isoproterenol, developed force at 4 Hz was equal to WT. Compared to normal levels, increased SERCA activity enhanced force development only at sub-physiological frequencies. A reduction in SERCA activity only showed depression of force at higher frequency range. Thus, generalizations regarding the correlation between SERCA activity and contractility can be highly ambiguous, because this relationship is critically dependent on factors including stimulation frequency.