The epinephrine-induced production of lactate in nonexercising muscles may be due in part to allosteric activation of 6-phosphofructo-1-kinase by fructose 2,6-bisphosphate (F-2,6-P2). To determine if a correlation exists between F-2,6-P2 and lactate production in skeletal muscle, isolated rat hindlimbs were perfused for 30 min with a medium containing epinephrine at concentrations varying between 1.7 +/- 0.5 and 72.4 +/- 4.2 nM. In comparison to control values, hindlimbs perfused with 72.4 +/- 4.2 nM epinephrine had a two- to threefold increase in F-2,6-P2 and a fourfold increase in muscle lactate production. Hindlimb lactate production was highly correlated to gastrocnemius adenosine 3′,5′-cyclic monophosphate (r = 0.80), fructose 6-phosphate (r = 0.87), and F-2,6-P2 (r = 0.81). The adenosine 3′,5′-cyclic monophosphate-mediated increase in glycogenolysis with consequent increase in fructose 6-phosphate (substrate for 6-phosphofructo-1-kinase and 6-phosphofructo-2-kinase) is likely important for induction of lactate production by inactive muscle. The high correlation between muscle F-2,6-P2 and muscle lactate production at varying concentrations of epinephrine supports the hypothesis that the epinephrine-induced activation of glycolysis and lactate production in nonexercising muscle is mediated in part by increases in F-2,6-P2 levels.