Highly purified sarcolemmal membranes were prepared from pig heart homogenates by differential and density gradient centrifugations. The membrane fragments exhibit ATP-dependent Ca2+-transport and Na+/Ca2+-exchange activities. ATP-dependent Ca2+-transport (K0.5Ca2+ = 0.3 microM; Vmax = 4.6 nmol Ca2+.mg protein-1.min-1) is not stimulated by oxalate. Ca2+-uptake is also not supported by p-nitro-phenylphosphate. Preincubation of sarcolemma with MgATP, calmodulin and catalytic subunit of cyclic AMP-dependent protein kinase stimulates active Ca2+-transport 1.8-fold. The effects of calmodulin and catalytic subunit are potentiating rather than additive. A large portion of the Ca2+ additionally accumulated after prephosphorylation of membranes is exchangeable for Na+ via the Na+/Ca2+-exchange system.