Inotropic interventions were compared with respect to their maximum effect on force of contraction in rat myocardium to identify limiting steps in calcium handling. Peak force, sarcomere length, and action potentials were measured in thin ventricular trabeculae. Relevant control conditions were stimulation frequency, 0.2 Hz; [Ca2+]o, 1 mM; [K+]o, 5 mM; [Na+]o, 150 mM. The inotropic interventions and results were as follows. 1) The interventions of high [Ca2+]o, low [Na+]o, high [K+]o, addition of tetraethylammonium chloride, or postextrasystolic potentiation resulted in approximately the same (within 5%) maximum force (Fmax). Above the respective optimum doses, force declined and aftercontractions were often observed. Combinations of the different interventions never enhanced force to above Fmax. This suggests that Fmax is determined by a maximum level of Ca2+ in the sarcoplasmic reticulum, above which spontaneous release occurs. 2) Sr2+ (10 mM) caused an increase of force to 1.3 X Fmax and lengthening of contraction and action potentials. The force-sarcomere length relation was, then, similar to that in skinned fibers at maximum activation. Hence, 1.3 X Fmax reflects saturation of the sarcomeres. We postulate that a large influx of Sr2+ during the long action potential can circumvent the reticulum and activate the sarcomeres directly. When the reticulum was blocked with ryanodine, maximum force of tetanic contractions was about 1.1 X Fmax. This result supports the above conclusions. 3) Isoproterenol increased force to a maximum that was 20% below Fmax and shortened the contraction. This may be due to a decreased sensitivity of the sarcomeres to Ca2+ or to stimulation of the Ca2+ pump in the reticulum, that is, an increasing fraction of the released Ca2+ is sequestered before it can activate the sarcomeres. Thus, three factors that limit force production were identified, depending on the inotropic stimulus.