The inhibitory G protein (Gi) has been proposed to participate in β2-adrenergic receptor (β2AR) signaling, but not in β1-adrenergic receptor (β1AR) signaling. Pertussis toxin (PTX) ADP-ribosylates and inhibits Gi, and recent data from the research group indicate that this leads to subsequent enhancement of β1AR signaling as well as direct adenylyl cyclase (AC) activation by forskolin. How this occurs if Gi is not coupled to the β1AR is not known. We hypothesize that the limited pool of Gβγ subunits in the cell is scavenged by the heterotrimeric PTX ADP-ribosylated Gi (Gαiβγ), forcing stimulatory G protein (Gs) into its more active, monomeric state, with a subsequent increase of cAMP accumulation and inotropic response. Alternatively, the PTX ADP-ribosylation can cause a shift in receptor-independent activation of Gi to Gs by nucleoside diphosphate kinases (NDPKs). Adult rat ventricular cardiomyocytes were transduced with an EGFP control virus or a Gβγ overexpressing virus ± PTX. The cells were then stimulated with noradrenaline in the presence of a PDE-inhibitor (IBMX) and a β2AR antagonist (ICI118551), and cAMP measured in a cAMP radioimmunoassay. A control experiment with a virus expressing the Gβγ-scavenger GRK2ct was performed similarly to the Gβγ experiment. Cells overexpressing NDPKs were incubated with PTX and stimulated with noradrenaline. Gβγ significantly reduced cAMP accumulation, possibly due to inhibitory effects on AC. Gβγ had a small tendency to lower the PTX-mediated enhancement of cAMP accumulation, this was however not significant. GRK2ct tended to increase cAMP accumulation, but no conclusion on its effect on the PTX response can be made due to experimental difficulties. NDPK did not have any effect on cAMP accumulation through the β1AR. Taken together, these studies do not support a role for Gβγ in mediation of PTX enhancement of β1AR- and forskolin-mediated cAMP accumulation. Although a role for Gβγ cannot be excluded, it seems more likely that PTX removes an intrinsic receptor independent constitutive inhibition of Gi upon AC. Future studies will be conducted to test this hypothesis. Furthermore, we did not find evidence to support a role for NDPKs in the PTX-mediated enhancement of the β1AR response, but NDPKs might be compartmented with the β2AR in rat cardiomyocytes.