Ablation of the enzyme phosphoinositide 3-kinase (PI3K) (PI3K^-/-) in mice increases cardiac contractility by elevating intracellular cAMP and enhancing sarcoplasmic reticulum Ca²⁺ handling. Because cAMP is a critical determinant of heart rate, we investigated whether heart rate is altered in mice lacking PI3K. Heart rate was similar in anesthetized PI3K^-/- and wild-type (PI3K^+/+) mice. However, IP injection of atropine (1 mg/kg), propranolol (1 mg/kg), or both drugs in combination unmasked elevated heart rates in PI3K^-/- mice, suggesting altered sinoatrial node (SAN) function. Indeed, spontaneous action potential frequency was 35% greater in SAN myocytes isolated from PI3K^-/- mice compared with PI3K^+/+ mice. These differences in action potential frequency were abolished by intracellular dialysis with the cAMP/protein kinase A antagonist Rp-cAMP but were unaffected by treatment with ryanodine to inhibit sarcoplasmic reticulum Ca²⁺ release. Voltage-clamp experiments demonstrated that elevated action potential frequencies in PI3K^-/- SAN myocytes were more strongly associated with cAMP-dependent increases in L-type Ca²⁺ current (I_Ca,L) than elevated hyperpolarization-activated current (I_f). In contrast, I_Ca,L was not increased in working atrial myocytes, suggesting distinct subcellular regulation of L-type Ca²⁺ channels by PI3K in the SAN compared with the working myocardium. In summary, PI3K regulates heart rate by the cAMP-dependent modulation of SAN function. The effects of PI3K ablation in the SAN are unique from those in the working myocardium.