We examined the relationships between PCr and Pi vs oxygen consumption or twitch rate during stimulation of rat muscle in situ and of perfused cat biceps and soleus muscles. In fast-twitch muscles such as rat gastrocnemius and cat biceps, we found a linear relationship between PCr and oxygen consumption. This result favors thermodynamic regulation of oxygen consumption by cytoplasmic phosphorylation potential rather than kinetic regulation by ADP. PCr changes during stimulation of rat muscle depleted of total creatine or adenine nucleotide were also not consistent with simple kinetic regulation by ADP. In cat soleus (slow-twitch) muscle, the observation of nonexponential PCr changes during stimulation suggested more complicated regulation, possibly involving changes in mitochondrial redox potential.