The effects of hypoxia on contractile tension and on tissue adenylate pool content, nicotinamide adenine nucleotide, NAD, nicotinamide adenine dinucleotide phosphate, NADP, and creatine phosphate, CrP, were investigated in isolated, spontaneously beating, guinea pig atria. When two different degrees of hypoxia were induced by lowering oxygen tension from 95% O2 (control) to 40% (moderate hypoxia) and 20% (severe hypoxia) for 30 min, contractile tension slowly decreased to 60% and 40% of control, respectively. In 40% O2 hypoxic atria, ATP was not significantly decreased, AMP slightly increased, TAN (total adenylate nucleotides) and adenylate energy charge [(ATP + 0.5 ADP)/(ATP + ADP + AMP)] did not change and creatine phosphate was decreased down to 53%. Hypoxic atria in 20% O2 showed a significant decrease of 26% in ATP, while ADP and AMP increased four and seven times, respectively. The adenylate energy-charge value was reduced from 0.93 to 0.70. Creatine phosphate decreased to below the analytical detection limit. Moderate hypoxia (40% O2), which induced a significant decrease of contractile tension but only minor changes of energetic tissue metabolism, was further investigated 2, 5, and 10 min after low oxygen tension was applied. Two stages of variations were evident during 30 min of experimental hypoxia. Within the first 10 min, concomitantly with atrial tension decrease, ATP, NAD, NADP, ATP/AMP, ATP/ADP, and TAN decreased, CrP began to decrease, inosine and xanthine showed no significant change. During the following 20 min of hypoxia, all parameters returned to the control levels with the exception of creatine phosphate. Adenylate energy charge did not change. The electrophysiological analysis of atrial cells did not show any major change in action potential configuration and resting potential, during 40% O2 hypoxia. The differences at metabolic level between moderate and more severe hypoxia suggest that the energetic state may be extremely unbalanced, in atrial tissue, as long as hypoxia is aggravated. Moreover, the time-course study, during 30 min of 40% O2, suggests that the early decrease of contractile tension does not depend on lowered energy availability, instead it might be, at least in part, a preventive measure to maintain energy balance in myocardial tissue to counteract hypoxic damage and, in this mechanism of defense, creatine phosphate shuttle seems to play a relevant role.