Smooth muscle Ca2+ handling is of major importance to understanding its function. A new approach utilizes molecular biology to develop transgenic mouse models in which the protein constituents of the various Ca2+ regulatory subsystems have been altered. Gene-targeted or gene-ablated (knockout) mice have been reported for the sarcoplasmic reticulum (SR) Ca2+ pump isoforms SERCA2, SERCA2a and SERCA3, the plasma membrane Ca2+ pump isoforms, PMCA1, PMCA2 and PMCA4, and the SR-associated protein, phospholamban (PLB), an inhibitor of SERCA2. A mouse line carrying a transgene for the smooth muscle specific expression of PLB has been reported. Evidence from studies using these mice combined with the classical pharmacological approaches has provided new insight into the relative role of the SR. We review this field with particular emphasis on PLB, since its modulation of SR function and smooth muscle contractility has the largest database. PLB via modulation of SERCA can play a major role in regulation of both phasic and tonic smooth muscle contractility. The use of transgenic mice has yielded surprises ,uch as PLB modulation of endothelial cell Ca2+ homeostasis, and the demonstration that PLB is the major site for A-kinase-mediated relaxation of mouse bladder. The use of these gene-altered models has provided evidence clearly implicating a major role for the SR in modulating smooth muscle Ca2+ and contractility, with the caveat that this modulation is tissue specific.