This study attempts to gain a better understanding of the primary cause of doxorubicin myotoxicity to aid in the development of a protocol to increase its effectiveness in treating muscle spasm diseases. The time course of acute injury to the orbicularis oculi muscle after injection of doxorubicin into the eyelids of rabbits was examined. The effects of doxorubicin injection were immediate and dramatic. Within 5 minutes, the muscle cells in the eyelids showed signs of myofibrillar dissolution. This process continued for the first 24 hours, with a marked decrease in the total number of myofibers by 1 hour after drug treatment. By 4 days after doxorubicin injection, most of the treated muscle had a more normal morphology, although some ongoing myofibrillar dissolution was present. There was evidence for a limited amount of muscle regeneration. Three levels of injury were seen: a severe, irreversible injury in the majority of the myofibers; a mild, reversible injury in a subset of the fibers; and a group of myofibers apparently uninjured as a result of doxorubicin treatment. Doxorubicin toxicity in skeletal muscle is of rapid onset. Understanding the primary cause and acute changes in doxorubicin myotoxicity may accelerate the development of improved protocols to increase the effectiveness of doxorubicin in treating muscle spasm diseases.