In heart failure, chronic catecholaminergic stimulation increases diastolic Ca²⁺ leak from ryanodine receptors (RyRs) of sarcoplasmic reticulum (SR), possibly due to the phosphorylation of RyRs through the activation of protein kinase A (PKA) or Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). In the present study, we attempted to identify which activated kinase is responsible for the enhanced Ca²⁺ leak caused by β-adrenergic stimulation. Trabeculae obtained from the hearts of adult male C57BL/6J mice were treated with isoproterenol and then permeabilized with saponin. To examine SR functions, Ca²⁺ in SR was released with caffeine and measured with fluo-3. The Ca²⁺ leak in isoproterenol-treated preparations was significantly increased when the PKA-dependent phosphorylation of RyR was increased without the involvement of CaMKII-dependent phosphorylation. Both the increase in Ca²⁺ leak and the phosphorylation of RyR were blocked by a PKA inhibitor. Our results show that β-adrenergic stimulation increases Ca²⁺ leak from SR through PKA-dependent phosphorylation of RyR.