Endothelial nitric-oxide synthase (eNOS) plays a crucial role in the regulation of a variety of cardiovascular and pulmonary functions in both normal and pathological conditions. Multiple signaling inputs, including calcium, caveolin-1, phosphorylation by several kinases, and binding to the 90-kDa heat shock protein (Hsp90), regulate eNOS activity. Here, we report a novel mechanism of G protein-dependent regulation of eNOS. We demonstrate that in mammalian cells, the α subunit of heterotrimeric G12 protein (Gα12) can form a complex with eNOS in an activation- and Hsp90-independent manner. Our data show that Gα12 does not affect eNOS-specific activity, but it strongly enhances total eNOS activity by increasing cellular levels of eNOS. Experiments using inhibition of protein or mRNA synthesis show that Gα12 increases the expression of eNOS by increasing half-life of both eNOS protein and eNOS mRNA. Small interfering RNA-mediated depletion of endogenous Gα12 decreases eNOS levels. A quantitative correlation can be detected between the extent of down-regulation of Gα12 and eNOS in endothelial cells after prolonged treatment with thrombin. G protein-dependent increase of eNOS expression represents a novel mechanism by which heterotrimeric G proteins can regulate the activity of downstream signaling molecules.