We describe the procedure for the measurement of intracellular calcium (Cai2+) simultaneously with function in the intact mouse heart and report findings in phospholamban-deficient mice. Seven phospholamban-deficient and six age-matched wild-type hearts were perfused retrogradely with oxygenated Krebs-Henseleit solution. Aequorin was injected into the apex of five hearts from each group to characterize Cai2+ transients. A pressure-sensing balloon was positioned in the left ventricle. Peak Cai2+ was significantly greater in the phospholamban-deficient hearts than in wild-type hearts (0.98 +/- 0.07 vs. 0.78 +/- 0.09 microM, p < 0.05). Calcium exchange was significantly faster in the phospholamban-deficient hearts. Cai2+ transients of significantly increased amplitude and decreased duration in phospholamban-deficient hearts correlated with increased contractility and faster relaxation. We report the first recordings of intracellular calcium transients in the intact heating mouse heart and present direct evidence that phospholamban is a key regulator of basal Cai2+ and contractility.