This study investigated cardiac excitation-contraction coupling at 37°C in transgenic mice with cardiac-specific overexpression of human β₂-adrenergic receptors (TG4 mice). In field-stimulated myocytes, contraction was significantly greater in TG4 compared with wild-type (WT) ventricular myocytes. In contrast, when duration of depolarization was controlled with rectangular voltage clamp steps, contraction amplitudes initiated by test steps were the same in WT and TG4 myocytes. When cells were voltage clamped with action potentials simulating TG4 and WT action potential configurations, contractions were greater with long TG4 action potentials and smaller with shorter WT action potentials, which suggests an important role for action potential configuration. Interestingly, peak amplitude of L-type Ca²⁺ current (I_Ca-L) initiated by rectangular test steps was reduced, although the voltage dependencies of contractions and currents were not altered. To explore the basis for the altered relation between contraction and I_Ca-L, Ca²⁺ concentrations were measured in myocytes loaded with fura 2. Diastolic concentrations of free Ca²⁺ and amplitudes of Ca²⁺ transients were similar in voltage-clamped myocytes from WT and TG4 mice. However, sarcoplasmic reticulum (SR) Ca²⁺ content assessed with the rapid application of caffeine was elevated in TG4 cells. Increased SR Ca²⁺ was accompanied by increased frequency and amplitudes of spontaneous Ca²⁺ sparks measured at 37°C with fluo 3. These observations suggest that the gain of Ca²⁺-induced Ca²⁺ release is increased in TG4 myocytes. Increased gain counteracts the effects of decreased amplitude of I_Ca-L in voltage-clamped myocytes and likely contributes to increased contraction amplitudes in field-stimulated TG4 myocytes.