The effects of the β-adrenergic receptor blocking agents propranolol and timolol on the initial calcium uptake velocity of sarcoplasmic reticulum vesicles of rabbit skeletal muscle were studied. Racemic d- and l-propranolol had similar inhibitory effects on initial calcium uptake velocity, which was inhibited 50% by 5–7 x 10-4 M racemic propranolol. Timolol was a much less potent inhibitor of initial calcium uptake velocity; 50% inhibition occurred at approximately 10-2 M timolol. Both drugs inhibited maximal calcium uptake velocity; however, Kca (the Ca2+ concentration at which calcium uptake was half-maximal) was modified differently. Propranolol increased Kca, whereas timolol caused the Kca to decrease. Addition of either drug to an ongoing calcium uptake reaction at the time that calcium content became maximal caused renewed calcium uptake. The relative potencies of propranolol and timolol as negative inotropic agents are similar to their potencies as inhibitors of sarcoplasmic reticulum calcium uptake, but dissimilar to their β-adrenergic receptor blocking potencies. Timolol, which has been reported to have less negative inotropic effect than propranolol, is approximately 5 times more potent than propranolol as a β-adrenergic receptor blocking agent but 15 times less potent as an inhibitor of sarcoplasmic reticulum calcium uptake. Inhibition of sarcoplasmic reticulum calcium uptake may thus characterize negative inotropic potencies of new β-adrenergic receptor blocking agents.