W e investigated the mechanisms responsible for altered contractile and relaxation function in overexpressed Gsα myocytes. Although baseline contractile function (percent contraction) in Gsα mice was similar to that of wild-type (WT) mice, left ventricular myocyte contraction, fura-2 Ca²⁺transients, and Ca²⁺ channel currents (I_Ca) were greater in Gsα mice in response to 10^–8 M isoproterenol (ISO) compared with WT mice. The late phase of relaxation of the isolated myocytes and fura-2 Ca²⁺ transients was accelerated at baseline in Gsα but did not increase further with ISO. In vivo measurements using echocardiography also demonstrated enhanced relaxation at baseline in Gsα mice. Forskolin and CaCl₂ increased contraction similarly in WT and Gsα mice. Rp-cAMP, an inhibitor of protein kinase, blocked the increases in contractile response and Ca²⁺ currents to ISO in WT and to forskolin in both WT and Gsα. It also blocked the accelerated relaxation in Gsα at baseline but not the contractile response to ISO in Gsα myocytes. Baseline measurements of cAMP and phospholambation phosphorylation were enhanced in Gsα compared with WT. These data indicate that overexpression of Gsα accelerates relaxation at end diastolic but does not affect baseline systolic function in isolated myocytes. However, the enhanced responses to sympathetic stimulation partly reflect increased Ca²⁺ channel activity; i.e the cellular mechanisms mediating these effects appear to involve a cAMP-independent as well as a cAMP-dependent pathway.