We compared mechanical activity and Ca2+ transients of ventricular myocytes isolated from wild-type and phospholamban (PLB)-deficient mouse hearts in control conditions and during beta-adrenergic stimulation. Compared with wild-type controls, cells isolated from PLB-deficient mouse hearts showed 1) a 2-fold increase in extent of cell shortening, 2) a 3-fold increase in maximal shortening velocity, and 3) a 3.4-fold increase in maximal relengthening velocity. PLB-deficient myocytes also demonstrated significant increases in the peak amplitude of the fura 2 fluorescence ratio and the rates of rising and falling phases of the Ca2+ transient. The fura 2 diastolic ratios were similar in both groups, suggesting no change in intracellular Ca2+ during diastole. In PLB-deficient myocytes, 0.05 microM isoproterenol induced an increase in the twitch amplitude by 152 +/- 11% (n = 6) compared with 290 +/- 31% (n = 6) in wild-type cells. Maximal shortening velocity was increased by 183 +/- 10% (n = 6) in PLB-deficient myocytes, compared with 398 +/- 62% (n = 6) in wild-type cells. The isoproterenol-induced increase in maximum relengthening velocity was increased by 168 +/- 8% (n = 6) in PLB-deficient cells compared with 445 +/- 71% (n = 6) in wild-type myocytes. In both groups, these changes in contractile parameters were accompanied by changes in the Ca2+ transient. Our results indicate that phosphorylation of sites other than PLB may play an important role in regulation of contraction-relaxation dynamics of heart cells responding to beta-adrenergic stimulation.