Phospholamban (PLB) regulates Ca²⁺- adenosinetriphosphatase activity in cardiac sarcoplasmic reticulum and participates in the regulation of myocardial performance. Animal models with altered levels of PLB permit in vivo evaluation of the physiological role of PLB. This study examined left ventricular (LV) performance in intact PLB heterozygous and homozygous mice under basal and stimulated conditions. A Millar Mikro-Tip transducer was inserted into the right carotid artery and advanced into the LV for direct measurement of ventricular pressure and the first derivative of intraventricular pressure (dP/dt). Baseline blood pressures were increased in PLB heterozygotes and even more so in PLB homozygotes compared with wild types (WT), and there were no differences in heart rate or LV end-diastolic pressure. The increase in pressure was primarily caused by an increase in systolic pressure. Baseline values for positive and negative dP/dt were linearly correlated with PLB levels. In PLB heterozygotes, contractile response to isoproterenol (Iso) was blunted compared with WT, but maximum rates of contraction were similar between the two groups. Contractile performance in PLB homozygous mice, which under baseline conditions was similar to maximum levels seen in WT, showed a blunted response to Iso, and maximum rates of contraction were significantly greater than in either of the other groups, indicating an essential but perhaps not exclusive role for PLB in mediating the inotropic effects of β-adrenergic agonists. The effects of Iso on negative dP/dt were also blunted in both PLB heterozygous and PLB homozygous animals. Our results demonstrate that myocardial function is highly dependent on PLB level and suggest that the cardiovascular effects of PLB perturbations are largely uncompensated for in the intact mouse.