The quantitative relationship between a given decrease in O2 tension (Po2) and the degree of depression of contractile function is difficult to define in intact myocardial tissues because of uncertainty in estimation of diffusion limitations. We therefore have studied the effects of hypoxia on contractility in a system with minimal series diffusion barriers, consisting of single cells within spontaneously contracting monolayers cultured from 10-day-old chick embryo ventricles. Reversible decreases in amplitude and velocity of cell wall motion occurred over a 3- to 4-min period when perfusate Po2 was less than 12 mmHg, and a maximum depression to about 30% of control levels occurred at Po2 values less than 4 mmHg. On reoxygenation, recovery of amplitude and velocity of contraction occurred in less than 15 s. Inhibition of glycolysis with 0.1 mM iodoacetate resulted in a more marked decrease in contraction amplitude and a significant slowing of beating rate during hypoxia. In addition, the critical Po2 at which contractile function became impaired was increased to about 16 mmHg. Thus, high-energy phosphates derived from anaerobic glycolysis appear to be able to support a reduced level of contractility during brief periods of hypoxia in these cultured heart cells.