The purine nucleotide cycle catalyzes the net reaction: aspartate + GTP + H2O----fumarate + NH3 + GFDP + Pi and leads to regeneration of AMP. In skeletal muscle the cycle's rate of operation increases several fold in response to a corresponding increase in work load, which results in a net increase in citric acid cycle intermediates and in release of ammonia. The same may be expected in heart muscle which, like skeletal muscle, possesses the enzymes of the purine nucleotide cycle. Isolated working rat hearts were therefore perfused for 45 min at low or high work load (0.16 v. 0.42 kg m/min/g dry wt) with glucose (5 mM) as substrate. Release of ammonia into the perfusate as well as the tissue content of citric acid cycle intermediates (citrate, isocitrate, 2-oxoglutarate, malate, and oxaloacetate), amino acids (aspartate, glutamate and glutamine), adenine nucleotides and phosphocreatine were measured in freeze-clamped tissue. There was no significant change in the sum of the citric acid cycle intermediates (1.295 v. 1.313 mumol/g dry wt), in aspartate (13.21 v. 14.32 mumol/g dry wt), in glutamate (15.58 v. 15.67 mumol/g dry wt), ATP (19.60 v. 19.17 mumol/g dry wt), ADP (5.00 v. 4.11 mumol/g dry wt), AMP (1.45 v. 1.01 mumol/g dry wt) and phosphocreatine (22.58 v. 25.80 mumol/g dry wt) when low and high work load were compared. Ammonia release was 26 mumol/h/g dry wt and 22 mumol/h/g dry wt at low and high work load respectively. The results suggest that in rat heart the activity of the purine nucleotide cycle does not increase with an increase in work load.(ABSTRACT TRUNCATED AT 250 WORDS)