Glucose-6-phosphate dehydrogenase (G6PD) activity of rat diaphragm muscle increased significantly 12 h after intrathoracic ('distal') denervation. Since this change represented one of the earliest known events after loss of innervation, the mechanism of neuronal regulation and the nature of the muscle response were further investigated. When, instead of distal denervation, the motor nerve was cut about 3.5 cm from the diaphragm, G6PD activity slightly decreased 12 h later. Denervations in which part of the same hemidiaphragm had a short nerve stump and another part, a long nerve stump, also showed a differential increase of G6PD activity dependent on length of nerve stump. In biochemical studies of the locus of the early post-denervation elevation of G6PD, enzyme activity was increased both in endplate and non-endplate regions of the muscle. Finally, the histochemical reaction for G6PD was more intense in both muscle fibers and interstitial elements, although the former made the major contribution to the biochemical results. In sum, muscle G6PD enzyme activity is closely regulated by a neurotrophic mechanism partly independent of nerve or muscle electrical activity.