Rats were fed on a diet containing the creatine analogue beta-guanidinopropionate (Gp) in order to deplete muscles of creatine. Diaphragm muscle from these rats contains about 80% less creatine and creatine phosphate (CrP) than normal; ATP levels are reduced about 45%. When stimulated with single shocks or brief tetani, the contractile response of diaphragm strips is very similar to normal with some slowing of relaxation. During a burst of intense activity (0.2-s tetanic stimulation every 0.5 s) the maximum tension, rate of tension development, and rate of relaxation all decrease rapidly to reach a minimum about 3 s from the onset of activity. In contrast, normal muscles show a small decrease in tension and relaxation rate but an increase in the rate of tension development under these conditions. No large decrease in ATP levels could be detected when the contractile parameters reached a minimum. Three possible mechanisms considered for the decline in contractile parameters are the following: (1) local regions of ATP depletion around the myofibrils; (2) a fall in the free energy of ATP hydrolysis (uG/d xi) due to inadequate buffering by CrP; and (3) decreased rate of proton utilization in the region of the myofibrils, where ATP turnover is most rapid. All three mechanisms may contribute to the observed effects, although the second seems the most likely to play a dominant role in modifying the contractile response.