The rate of biosynthesis of pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) was compared in cells maintained under normoxic or hypoxic conditions. L8 cells (a myoblast cell line) were pulse-labeled with [3H]leucine and incorporation of radioactivity into pyruvate kinase was measured after quantitative affinity separation with anti-pyruvate kinase monoclonal antibody. During chronic hypoxia there is an increased rate of biosynthesis of pyruvate kinase leading to an increase in enzyme content and augmented glycolytic capacity. An inhibitor of the electron transport chain, antimycin A, was used to determine whether changes in pyruvate kinase content occurring during hypoxia are a result of reduction in molecular oxygen directly or an indirect consequence of oxygen depletion. Pyruvate kinase activity increased during chronic antimycin A exposure under normoxic conditions. The increase was quantitatively accounted for by an increase in cellular pyruvate kinase enzyme content. This suggested that decreases in the levels of molecular O2 are not the direct stimulus for the increased content of pyruvate kinase. It is more likely that the increased pyruvate kinase content results from depressed rates of electron transport through the mitochondrial electron transport chain.