Experiments are reported demonstrating that differential rates of inactivation of the histochemical staining for myofibrillar actomyosin ATPase in rat skeletal muscle fibers exist following inclusion of low concentrations of Cu2+ in the preincubation medium. This response of rat muscle occurs at near neutral (7.40), acid (4.60), and alkaline (10.30) pH. The response to Cu2+ appears to result from a binding of Cu2+ onto the myofibrillar complex, probably on myosin itself, as it can be reversed by soaking of the pretreated muscle sections in sodium cyanide or the Cu2+ chelator diethyldithiocarbamate. The pattern of modification of the staining pattern following pretreatment with Cu2+ is the mirror image of that produced by pretreatment with acid. The results demonstrate that the inclusion of Cu2+ in the preincubation media for the myofibrillar actomyosin ATPase can be a useful tool to differentiate fiber types. They also support the earlier conclusion that three distinct types of type II fibers can be identified in rat skeletal muscle based on the histochemical staining for myofibrillar actomyosin ATPase.