Phospholamban (PLB) is a protein associated with the Ca(2+)-ATPase of the sarcoplasmic reticulum (SR) in cardiac, slow-twitch skeletal, and smooth muscle. PLB inhibits the SR Ca(2+)-ATPase in cardiac muscle; this inhibition is relieved on phosphorylation. The role of PLB in smooth muscle contractility is less clear. To elucidate the role of PLB in vascular smooth muscle contractility in vivo, we used a model in which the PLB gene was targeted in murine embryonic stem cells, generating mice deficient in PLB (PLB-). The PLB- mice exhibited no gross developmental abnormalities, but marked changes in aortic contractility were observed. The time course of force development with phenylephrine stimulation was faster in the PLB- aorta, suggesting changes in SR Ca2+ release. No differences were observed for KCl contractures between tissue types for either maximum forces observed or time course of force production; relaxation was faster in 7 of 11 arteries, but this trend did not attain statistical significance. The cumulative concentration-isometric force relations for the PLB- aorta were to the right of the wild-type for both KCl and phenylephrine stimulation, indicating a less sensitive tissue. To investigate whether the observed changes were related to SR function, we inhibited the SR Ca(2+)-ATPase with cyclopiazonic acid (CPA). CPA treatment resulted in a leftward shift of the concentration-isometric force relations for both aorta types, as expected after removal of a major Ca2+ uptake system. Most interestingly, the differences between PLB and wild-type aorta were abolished by SR inhibition. Our results suggest that PLB is a regulator of the SR Ca2+ pump in mouse aorta and plays a regulatory role in both KCl-induced and receptor-mediated contractility in vascular smooth muscle.