An increased phospholamban (PLB)-to-sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) ratio has been suggested to contribute to the slowing of relaxation in failing human ventricle. We have used an adenoviral vector carrying the sequence for PLB to increase this ratio in isolated adult rat ventricular myocytes, and we have examined the functional consequences. With use of adenoviral vectors, the PLB content of adult rat myocytes was increased 2.73-fold, with SERCA2a levels unchanged. Maximum contraction amplitude of PLB-overexpressing myocytes was decreased to 6.9 +/- 0.3% shortening compared with 11.2 +/- 0.8% for 24-h controls (Con; P < 0.001, 5 preparations, 103 myocytes). Maximum rates of shortening and relengthening were also significantly decreased. Ca(2+) transient amplitudes were slightly depressed, and time to 50% decay of the transients was significantly increased: 237 +/- 18 (n = 14 myocytes) and 432 +/- 32 ms in Con and PLB (n = 15) myocytes, respectively (P < 0.001). The amount of Ca(2+) in the sarcoplasmic reticulum stores was reduced by 21% (P < 0.05). Relaxation was significantly slower in PLB than in Con myocytes when the Na(+)/Ca(2+) exchanger was blocked but not when sarcoplasmic reticulum Ca(2+) uptake was inhibited. Adenovirus infection with Ad.RSV.PLB was therefore able to produce functional changes in adult cardiac myocytes within 24 h, consistent with overexpression of PLB and similar to those seen in failing human heart.