Rats were fed on a diet containing 1% beta-guanidinopropionate (Gp) to deplete their muscles of creatine. The apparent energy reserves (creatine phosphate (CrP) + ATP) of rested state diaphragm muscle strips were found to be 79% depleted by this treatment. To determine if the effective energy reserves for contraction were depleted to a similar extent, the response to direct electrical stimulation (0.2-s tetani) was measured in the presence of inhibitors of respiration (NaCN) and glycolysis (iodoacetate). Only 4 +/- 1 contractions could be elicited from strips from Gp-fed animals. Normal strips gave 15 +/- 2 contractions under the same conditions. For both sets of diaphragms the energetic cost of contraction in terms of approximately P was approximately 1 mumol/g wet weight. The mean level of Pi generated following stimulation to exhaustion was 10.1 mumol/g more in normal than in depleted strips. It is concluded that no significant additional energy stores such as phosphorylated Gp are readily available for contraction in muscles depleted of creatine by Gp treatment.