Previous work suggested that altered Ca2+ homeostasis might contribute to dysfunction of nebulinfree muscle, as gene expression analysis revealed that the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-inhibitor sarcolipin (SLN) is up-regulated >70-fold in nebulin knockout mice, and here we tested this proposal. We investigated SLN protein expression in nebulin-free and wild-type skeletal muscle, as well as expression of other Ca2+-handling proteins. Ca2+ uptake capacity was determined in isolated sarcoplasmic reticulum vesicles and in intact myofibers by measuring Ca2+ transients. Muscle contractile performance was determined in skinned muscle activated with exogenous Ca2+, as well as in electrically stimulated intact muscle. We found profound up-regulation of SLN protein in nebulin-free skeletal muscle, whereas expression of other Ca 2+-handling proteins was not (calsequestrin and phospholamban) or was minimally (SERCA) affected. Speed of Ca2+ uptake was >3-fold decreased in sarcoplasmic reticulum vesicles isolated from nebulin-free muscle as well as in nebulin-free intact myofibers. Ca2+-activated stress in skinned muscle and stress produced by intact nebulin-free muscle were reduced to a similar extent compared with wild type. Half-relaxation time was significantly longer in nebulin-free compared with wild-type muscle. Thus, the present study demonstrates for the first time that nebulin might also be involved in physiological Ca2+ handling of the SR-myofibrillar system.