Previous work suggested that altered Ca²⁺ homeostasis might contribute to dysfunction of nebulin-free 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 Ca²⁺-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.—Ottenheijm, C. A. C., Fong, C., Vangheluwe, P., Wuytack, F., Babu, G. J., Periasamy, M., Witt, C. C., Labeit, S., Granzier, H. Sarcoplasmic reticulum calcium uptake and speed of relaxation are depressed in nebulin-free skeletal muscle.