The effects of phosphorylation status on Ca²⁺release and Ca²⁺ removal were studied in fast-twitch flexor digitorum brevis and slow-twitch soleus skeletal muscle fibers enzymatically isolated from wild-type and phospholamban knockout (PLBko) mice. In all fibers the adenosine 3′,5′-cyclic monophosphate-dependent protein kinase (PKA) inhibitor H-89 decreased the peak amplitude of the intracellular Ca²⁺ concentration ([Ca²⁺]) transient for a single action potential, and the PKA activator dibutyryl adenosine 3′,5′-cyclic monophosphate (DBcAMP) reversed this effect, indicating modulation of Ca²⁺release by phosphorylation status in all fibers. H-89 decreased the decay rate constant of the [Ca²⁺] transient and DBcAMP reversed this effect only in phospholamban-expressing fibers (wild-type soleus), indicating modulation of Ca²⁺ removal only in the presence of phospholamban. A high basal level of PKA phosphorylation in soleus fibers maintained under our control conditions was indicated by the lack of effect of direct application of DBcAMP on Ca²⁺ release or Ca²⁺ removal in wild-type or PLBko soleus fibers and was confirmed by analysis of phospholamban from wild-type soleus fibers.