Muscle fibers of rat gastrocnemii were punctured in vivo with a tungsten wire whose tip was 10 microns wide. After two hours, most punctured fibers had clearly necrotic segments. The length of necrotic segments appeared to increase between three and four hours. Demarcation of surviving stumps from necrotic segments by a membrane was complete in most muscles by seven hours. At the boundary between necrotic segments and nondemarcated stumps there was a cap of densely aggregated or hypercontracted myofilaments which stained strongly for precipitated calcium. The membrane which demarcated the stumps was found partially formed in the necrotic pulp close to this cap, attached to the plasma membrane. While forming it had a free edge and probably resulted from spontaneous aggregation of phospholipids in the pulp. Other portions of newly formed membrane were found encircling organelles inside necrotic segments. Prompt formation of a demarcating membrane probably limits the extent of necrosis in injured fibers.