Two-dimensional phosphorus chemical shift imaging (2D-31P-CSI) was used to investigate macroscopic heterogeneity within the flexor digitorum profundus (FDP) muscle of the human forearm during exercise. Subjects performed low-frequency steady-state finger flexion exercise at submaximal work levels using a bulb ergometer. The number of fingers actively involved in finger flexion exercise was varied in a total of 12 experiments. Active muscles could be determined by the increase of Pi and decrease of phosphocreatine observed in the localized spectra. Within the FDP, regions of active and inactive fibers were significantly different in levels of Pi and phosphocreatine (P less than 0.01) during flexion of individual fingers. Individual flexion of the index finger was found to involve only fibers in the deep region of the FDP. Fibers in the central region were found to be involved in flexion of the middle finger, and fibers in the superficial region were involved in flexion of the ring and little fingers. The results of this study demonstrate the potential of 2D-31P-CSI for in vivo investigation of intramuscular heterogeneity in human skeletal muscle.