The aim of this work was to study whether changes in fructose 2,6-bisphosphate concentration are correlated with variations of the glycolytic flux in the isolated working rat heart. Glycolysis was stimulated to different extents by increasing the concentration of glucose, increasing the workload, or by the addition of insulin. The glycolytic flux was measured by the rate of detritiation of [2-3H]- and [3-3H]glucose. Under all the conditions tested, an increase in fructose 2,6-bisphosphate content was observed. The glucose- or insulin-induced increase in fructose 2,6-bisphosphate content was related to an increase in the concentration of fructose 6-phosphate, the substrate of 6-phosphofructo-2-kinase. An increase in the workload correlated with a 50% decrease in the Km of 6-phosphofructo-2-kinase for fructose 6-phosphate. Similar changes in Km have been observed when purified heart 6-phosphofructo-2-kinase was phosphorylated in vitro by the cyclic AMP-dependent protein kinase or by the calcium/calmodulin-dependent protein kinase. Since the concentration of cyclic AMP was not affected by increasing the workload, it is possible that the change in Km of 6-phosphofructo-2-kinase, which was found in hearts submitted to a high load, resulted from phosphorylation by calcium/calmodulin protein kinase; other possibilities are not excluded. Anoxia decreased the external work developed by the heart, stimulated glycolysis and glycogenolysis, but did not increase fructose 2,6-bisphosphate.