Diaphragmatic function and biochemical changes were studied during respiratory failure induced by incremental inspiratory threshold loading in anesthetized rabbits (1) who were unbound and spontaneously breathing, (2) during lower thoracic and abdominal binding, and (3) while bound and undergoing transvenous phrenic nerve pacing of the diaphragm. There was no evidence of contractile fatigue or alterations in glycogen or lactate concentrations in the diaphragm of unbound spontaneously breathing animals. With thoracoabdominal binding, there was a fall in maximal transdiaphragmatic pressure (Pdimax) and the ratio of diaphragmatic force divided by neural input (Pdi/Edi ratio); there was no change in diaphragm glycogen, but there was a significant rise in lactate. In the bound and phrenic-paced animals Pdimax and Pdi/Edi ratio fell, and there was significant glycogen depletion and lactate accumulation. There was a significant correlation between diaphragm function and the levels of diaphragm glycogen and lactate at the point of respiratory failure. We conclude that (1) respiratory failure induced by incremental inspiratory threshold loading was not associated with either contractile fatigue of the diaphragm or diaphragmatic biochemical changes in unbound spontaneously breathing animals, (2) when mechanisms that preserve diaphragmatic function are circumvented by phrenic pacing and/or thoracoabdominal binding, diaphragm fatigue and biochemical changes occur, and (3) there is a significant relationship between in vivo evidence of contractile fatigue of the diaphragm and diaphragmatic glycogen depletion and lactate accumulation.