A method has been developed for separating slow- and fast-twitch fibres from the same muscle for analysis of high-energy phosphate compounds by an enzymatic fluorometric technique. Since the ATP and creatine phosphate concentrations in single muscle fibres are at pmol levels, enzymatic cycling is necessary to measure the NADPH formed in the reaction. The precision of the method has been found to be 3.3% for ATP and 2.0% for creatine phosphate. Variations in the levels of high-energy phosphate compounds were no greater between fibres of the same type than regionally within a single fibre. When slow- and fast-twitch fibres were compared there were no significant differences in the high-energy phosphate concentrations, except that in women there was significantly more creatine phosphate in slow-twitch than in fast-twitch fibres.