In organ culture of rat diaphragm, the presence of a 2-2.5 cm phrenic nerve stump delays the time of failure of miniature endplate potentials and eliminates the increase in glucose-6-phosphate dehydrogenase (G6PD) activity which otherwise occurs at 16.5 h in muscles cultured without nerve stumps. The nerve stump effect persists in the presence of blocking doses of D-tubocurarine but is eliminated by nerve crush. As shown by studies of amino acid incorporation into protein, the effect does not involved an overall change in protein synthesis. Effluents collected over 1-2 h from unstimulated or stimulated phrenic nerve-muscle preparations had no effect on G6PD activity when applied to muscles cultured without nerve stumps. However, medium conditioned by use in organ cultures with long nerve stumps partially countered the denervation-like effect in host cultures. Thus, the nerve maintains muscle G6PD by a humoral mechanism probably unrelated to impulse activity or nicotinic receptor activation.