Early studies showed that in addition to GTP, the pyrimidine nucleotides UTP and CTP support activation of the adenylyl cyclase (AC)-stimulating Gs protein. The aim of this study was to elucidate the mechanism by which UTP and CTP support Gs activation. As models, we used S49 wild-type lymphoma cells, representing a physiologically relevant system in which the β2-adreno-receptor (β2AR) couples to Gs, and Sf9 insect cell membranes expressing β2AR-Gαs fusion proteins. Fusion proteins provide a higher sensitivity for the analysis of β2AR-Gs coupling than native systems. Nucleoside 5′-triphosphates (NTPs) supported agonist-stimulated AC activity in the two systems and basal AC activity in membranes from cholera toxin-treated S49 cells in the order of efficacy GTP ≥ UTP > CTP > ATP (ineffective). NTPs disrupted high affinity agonist binding in β2AR-Gαs in the order of efficacy GTP > UTP > CTP > ATP (ineffective). In contrast, the order of efficacy of NTPs as substrates for nucleoside diphosphokinase, catalyzing the formation of GTP from GDP and NTP was ATP ≥ UTP ≥ CTP ≥ GTP. NTPs inhibited β2AR-Gαs-catalyzed [γ-32P]GTP hydrolysis in the order of potency GTP > UTP > CTP. Molecular dynamics simulations revealed that UTP is accommodated more easily within the binding pocket of Gαs than CTP. Collectively, our data indicate that GTP, UTP, and CTP interact differentially with Gs proteins and that transphosphorylation of GDP to GTP is not involved in this G protein activation. In certain cell systems, intracellular UTP and CTP concentrations reach ∼10 nmol/mg of protein and are higher than intracellular GTP concentrations, indicating that G protein activation by UTP and CTP can occur physiologically. G protein activation by UTP and CTP could be of particular importance in pathological conditions such as cholera and Lesch-Nyhan syndrome.