Abstract The drug, 20,25-diazacholesterol (20,25-DC; 300 mg/kg), was administered intragastricly to male Wistar rats on days 0 and 7. At various times afterward the mechanical and electrical properties of the fast-twich extensor digitorum longus (extensor) and the slow-twitch soleus muscles were evaluated. In vivo recordings revealed that at 16 to 35 days after beginning the 20,25-DC treatment the neurally evoked twitch of the extensor muscle had a relaxation time which was 16 to 19 times that of control. This abnormality was associated with an increase of posttetanic potentiation and, in some muscles, a brief contracture. These mechanical abnormalities disappeared as the extensor was subjected to continuous activity or “warmup.” In marked contrast to the extensor, treatment with 20,25-DC did not alter the mechanical properties of the soleus. Furthermore, 41% of the fibers in the experimental extensor produced trains of action potentials after a single direct stimulus, whereas such activity was found in only 2% of the corresponding soleus fibers. Thus, a differential sensitivity of these muscles to the myotonia-inducing effect of 20,25-DC was established. A detailed analysis of the passive cable properties of the sarcolemma revealed that the chloride conductance was reduced to approximately 30% of control for both experimental muscles. Likewise, 20,25-DC caused an equivalent increase of desmosterol and decrease of cholesterol content of the extensor and soleus muscles. Therefore, the distinct functional changes of the extensor muscle seen in this study cannot be due to a differential effect of 20,25-DC on sterol content or chloride permeability of the sarcolemma. The data support the hypothesis that inherent differences in the action potential mechanism predispose the fast muscle to 20,25-DC-induced myotonia.