Phosphorus nuclear magnetic resonance (NMR) spectra and twitch tension were recorded during stimulation of gastrocnemius muscles of pentobarbital sodium-anesthetized rats which had been fed the creatine analogue beta-guanidinopropionic acid (beta-GPA, 2% diet) for periods from 0 (control) to 8 wk. Total creatine content of unstimulated muscles decreased by 42, 67, 82, and 88% compared with controls after 2-, 4-, 6-, and 8-wk feeding, respectively. The staircase effect observed in control muscles during 8 min of twitch stimulation at 0.25, 0.5, and 0.75 Hz was reduced after 2- to 8-wk beta-GPA feeding. However, after 6- to 8-wk feeding, the twitch force at the end of 8 min of stimulation was not different from controls. The time constant for phosphocreatine (PCr) changes at the onset of and during recovery after stimulation was proportional to total creatine content. The relationship between PCr content and twitch rate times force at the end of stimulation was linear, with slope proportional to total creatine content. PCr content in beta-GPA-fed animals was transiently greater during recovery than before stimulation, suggesting a regulatory effect of the inorganic phosphate released by hydrolysis of phosphorylated beta-GPA. The results are consistent with linear models of respiratory control in which the creatine kinase reaction acts as a simple buffer of adenine nucleotide levels.