The rates of 14carbon incorporation into CO2 and glycogen from [U-14C]-lactate and [1-14C]acetate in frog sartorius muscles were compared. The rates of incorporation into CO2 were similar, while the rate of incorporation of lactate into glycogen was more than 200-fold larger than that of acetate incorporation. It is concluded that the pathway of lactate incorporation into glycogen does not involve Krebs cycle intermediates and is extramitochondrial. To test the possibility that the pathway of lactate incorporation involves net reversal of a pyruvate kinase, the changes in phosphoenolpyruvate and pyruvate concentrations during stimulation of lactate incorporation into glycogen were measured. There wer none. The mass action ratio of pyruvate kinase was calculated. This value was two orders of magnitude from the equilibrium constant and it was concluded that reversal of pyruvate kinase was a very unlikely pathway. To test the possibility that a pathway involving the oxaloacetate-to-phosphoenolpyruvate step was involved the muscles were treated with 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase. The treatment resulted in decreased incorporation of lactate into glycogen.