The correlation between phospholamban and sarcoplasmic reticulum Ca(2+)-transporting ATPase levels and the magnitude of phospholamban-mediated stimulation of sarcoplasmic reticulum Ca2+ transport was examined in microsomes prepared from rabbit and canine cardiac, slow twitch and fast twitch skeletal muscle. Phospholamban was absent from microsomes prepared from fast twitch skeletal muscle but present at comparable levels in microsomes prepared from cardiac and slow twitch skeletal muscle. Levels of Ca(2+)-transporting ATPase were higher in microsomes prepared from slow twitch skeletal muscle than in microsomes prepared from cardiac muscle, however, and ratios of phospholamban to Ca(2+)-transporting ATPase were several fold greater in microsomes prepared from cardiac muscle than in microsomes prepared from slow twitch skeletal muscle. Stimulation of ATP-dependent Ca2+ transport following phosphorylation of phospholamban by cAMP-dependent protein kinase or incubation with anti-phospholamban monoclonal antibody was observed only in cardiac muscle microsomes. These observations indicate that phospholamban, while present in both cardiac and slow twitch skeletal muscle, may be involved in the hormonal regulation of sarcoplasmic reticulum Ca2+ transport only in the former, and that the lack of phospholamban-mediated stimulation of Ca2+ transport in slow twitch skeletal muscle sarcoplasmic reticulum may result from the lower ratio of phospholamban to Ca(2+)-transporting ATPase in this tissue.