The role of creatine kinase (CK) bound to sarcoplasmic reticulum (SR), in the energy supply of SR ATPase in situ, was studied in saponin-permeabilised rat ventricular fibres by loading SR at pCa 6. 5 for different times and under different energy supply conditions. Release of Ca2+ was induced by 5 mM caffeine and the peak of relative tension (T/Tmax) and the area under isometric tension curves, ST, were measured. Taking advantage of close localisation of myofibrils and SR, free [Ca2+] in the fibres during the release was estimated using steady state [Ca2+]/tension relationship. Peak [Ca2+] and integral of free Ca2+ transients (S[Ca2+]f) were then calculated. At all times, loading with 0.25 mM adenosine diphosphate, Mg2+ salt (MgADP) and 12 mM phosphocreatine (PCr) [when adenosine triphosphate (ATP) was generated via bound CK] was as efficient as loading with both 3.16 mM MgATP and 12 mM PCr (control conditions). However, when loading was supported by MgATP alone (3.16 mM), T/Tmax was only 40% and S[Ca2+]f 31% of control (P < 0.001). Under these conditions, addition of a soluble ATP-regenerating system (pyruvate kinase and phosphoenolpyruvate), did not increase loading substantially. Both ST and S[Ca2+]f were more sensitive to the loading conditions than T/Tmax and peak [Ca2+]. The data suggest that Ca2+ uptake by the SR in situ depends on local ATP/ADP ratio which is effectively controlled by bound CK.