Skeletal muscle glucose uptake closely reflects muscle activity at exercise intensity levels <55% of maximal oxygen consumption (VO2max). Our purpose was to evaluate individual skeletal muscle activity from glucose uptake in humans during pedaling exercise at different workloads by using [18F]fluorodeoxyglucose (FDG) and positron emission tomography (PET). Twenty healthy male subjects were divided into two groups (7 exercise subjects and 13 control subjects). Exercise subjects were studied during 35 min of pedaling exercise at 40 and 55% VO2max exercise intensities. FDG was injected 10 min after the start of exercise or after 20 min of rest. PET scanning of the whole body was conducted after completion of the exercise or rest period. In exercise subjects, mean FDG uptake [standardized uptake ratio (SUR)] of the iliacus muscle and muscles of the anterior part of the thigh was significantly greater than uptake in muscles of control subjects. At 55% VO2max exercise, SURs of the iliacus muscle and thigh muscles, except for the rectus femoris, increased significantly compared with SURs at 40% VO2max exercise. Our results are the first to clarify that the iliacus muscle, as well as the muscles of the anterior thigh, is the prime muscle used during pedaling exercise. In addition, the iliacus muscle and all muscles in the thigh, except for the rectus femoris, contribute when the workload of the pedaling exercise increases from 40 to 55% VO2max.