The essential conditions for the Ca2+ releasing action of caffeine from isolated sarcoplasmic reticulum (SR) of rabbits were evaluated by an investigation into the effects of Ca2+, Mg2+, MgATP2-, and ATP concentration, ionic strength, and degree of loading. The heavy fraction (4,500×g) of the reticulum was used. Except for the study on degree of loading, 0.2 mg protein·ml-1 SR was loaded actively with 0.02 mM45CaCl2, resulting in >90 nmol·mg protein-1 at steady state, and then the effects of various parameters with or without (control) caffeine were tested.