Mitochondrial respiration/oxidative phosphorylation is the main source of energy, in the form of ATP, in the heart under physiological conditions. Different respiratory substrates were used in various experiments during heart perfusion: glucose, pyruvate, lactate, glucose+pyruvate, glucose+lactate, glucose+insulin etc. Also under physiological conditions, the concentration of respiratory substrates/hormones in blood can vary significantly. In the present study, we tested the effect of pyruvate, lactate and insulin (all in the presence of glucose) and glucose (in the presence of pyruvate) on the ATP-producing and -consuming blocks in perfused rat heart, in a system where HR (heart rate) was allowed to vary (no pacing). Changes in RPP (rate-pressure product) and PCr (phosphocreatine) concentration were measured. PAA (Proportional Activation Approach) was used to visualize and quantitatively analyse the data. It was demonstrated that addition of glucose (in the presence of pyruvate) exerted essentially no effect on the system. Insulin (in the presence of glucose) activated only the ATP producer. The most interesting finding is that, in our system, pyruvate and lactate (added in the presence or instead of glucose) activated ATP producer, but significantly inhibited ATP consumer (their effect was quantitatively identical).