Multiple isoforms of inhibitory G-subunits (G_i1,2,3 as well as G_o) are present within the heart and their role in modulating pacemaker function remains unresolved. Do inhibitory G-subunits selectively modulate parasympathetic heart rate responses? Published findings using a variety of experimental approaches have implicated roles for G_i2, G_i3 and G_o in parasympathetic signal transduction. We have compared in vivo different groups of mice with global genetic deletion of G_i1/G_i3, G_i2 and G_o against littermate controls using implanted ECG telemetry. Significant resting tachycardia was observed in G_i2 (-/-) and Go (-/-) mice compared with control and Gi1/G_i3 (-/-)(-/-) mice (p-value <0.05). Loss of diurnal heart rate variation was seen exclusively in G_o (-/-) mice. Using heart rate variability (HRV) analysis, compared to littermate controls (4.02ms±21.17(n=6), G_i2 (-/-) mice have a selective attenuation of HF power (0.73ms²±0.31(n=5)), p-value<0.05). G_i1/G_i3 (-/-)(-/-) and G_o (-/-) cohorts have non-significant changes in HF power. G_o (-/-) mice have a different basal HRV signature. The observed HRV phenotype in G_i2 (-/-) mice was qualitatively similar to atropine (1mg/kg) treated controls (and mice treated with the GIRK channel blocker tertiapinQ (0.05mg/kg)). Maximal cardio-inhibitory response to the M2-receptor agonist carbachol (0.5mg/kg) compared to basal heart rate was attenuated in G_i2 (-/-) mice (0.08+/-0.04(n=6)) compares to control (0.27+/-0.04 (n=7) p-value<0.05). Our data suggests a selective defect of parasympathetic heart rate modulation in mice with G_i2 deletion. Mice with G_o deletion also have a defect in short term heart rate dynamics but this is qualitatively different to the effects of atropine, tertiapinQ and G_i2 deletion. In contrast G_i1 and G_i3 do not appear to be essential for parasympathetic responses in vivo.