The specific binding of 125I-insulin by rat soleus muscle was depressed when muscle ATP was depleted, either by prolonged anoxia or more rapidly with 2,4-dinitrophenol. Insulin binding was not eliminated in ATP-depleted muscle, but was reduced by 70--80%. Insulin binding by aerobic muscle could be resolved into two components; a high-affinity, low-capacity site (KD = 7.8 nM) and a low-affinity, high-capacity site (KD = 390 nM). The stimulatory effect of insulin on xylose uptake could be correlated with binding to the high-affinity site. These results indicate that there is some ATP-dependent process involved in the regulation of insulin binding by soleus muscle. It is suggested that this could be a phosphorylation-dephosphorylation system, acting either on the receptor itself or on some closely related membrane protein.