The fast-twitch extensor digitorum longus and the slow-twitch soleus muscles of the rat were incubated for 1 to 6 hours in vitro in oxygen-free medium or a medium containing potassium cyanide. Within 3 hours of incubation, characteristic structural alterations of the sarcoplasmic reticulum were observed in extensor digitorum longus muscle fibers. Concomitant with the disruption of the normal sarcotubular organization there was a selective clustering of sarcoplasmic reticulum elements with formation of tubular aggregates similar to those observed in human muscle under various pathologic conditions. The aggregates increased in size by apposition of new tubules at the periphery and acquired a regular hexagonal arrangement. Double-walled tubules, apparently derived from folding of sarcoplasmic reticulum tubules, were also contained in the aggregates. Electron-opaque material was seen connecting adjacent tubules. It is proposed that this material derives from the Z-bands which appeared largely extracted since early stages of anoxic injury. Tubular aggregates were not seen in the soleum muscle fibers incubated under the same conditions in vitro. The results indicate that tubular aggregates can be formed by simple rearrangement of the sarcoplasmic reticulum without proliferation of new sarcotubular elements.