We investigated whether hypoxia altered the utilization of fructose-1,6-bisphosphate as a gluconeogenic or glycolytic intermediate in superfused media from hog carotid artery. Using 13C-NMR, we found that although 3-13C-iactate production from l-13C-glucose increased compared to that under well-oxygenated conditions, the conversion of exogenously applied l,6-13C-fructose-l,6-bisphosphate to glucose (gluconeogenesis) or to 3-13C-lactate was not significantly affected by hypoxia. Since hypoxia alters the rate of glucose conversion to lactate but not the conversion of fructose-1,6-bisphosphate to glucose, we conclude that glycolysis and glycogenolysis may continue to be compartmentalized during hypoxia and that a high rate of gluconeogenesis can occur even during hypoxia.