Acetylcholine released from the vagus nerve acts on cardiac M2 receptors to cause negative chronotropic responses and this is due, in part to an increase in K⁺ conductance mediated by G-protein inwardly rectifying potassium (GIRK)channels (Sakmann et al. 1983. This process is pertussis toxin sensitive implicating Gi/o in GIRK channel activation. Ablation of cardiac GIRK channels at a molecular level leads to impaired parasympathetic responses in vivo (Wickman et al. 1994). Giα2 is considered the most abundant cardiac isoform. We screened mice on an Sv129 background with global genetic deletions of Gαi2 and Gαi 1 and 3 as a double knockout with littermate controls (adjusted for age, weight and sex) and assessed negative chronotropic responses to intraperitoneal carbachol administration (500ng/g) under inhalation isoflurane anaethesia. Maximal relative inhibitory response was attenuated in Gαi2-deficient mice (mean inhibition 0.1152 ± 0.024, n=6) compared to control (0.3261 ± 0.054, n=6) or Gαi1/3 (0.3280 ± 0.080, n=6). Additionally, using heart rate variability analysis (HRV) of ECG data collected from implantable telemetry devices we were able to measure heart rate dynamics in conscious freely moving mice (Gehrmann et al. 2000). Our results suggest differential heart rate responses with respect to particular Giα isoform. Mean heart rate (HR) over 48hrs demonstrated that Gαi2 (-/-) deficient mice had significantly higher nocturnal HR (Gαi2, 617.6 ± 11.89 (n=5) vs control, 568.5 ± 14.64 (n=6) vs Gαi1/3, 579 ± 8.2 (n=6) (1-way ANOVA, P < 0.05, n=6). Similarly day time heart rate showed a similar trend. We also examined HR dynamics in the short term in both frequency and time domains. Total power (TP), low frequency (LF) and high frequency (HF) spectral components of power were significantly reduced with respect to littermate controls and Gαi1/3 double knockouts (1-way ANOVA, P <0.05, n=6). Similarly in the time domain mean N-N interval was signifcantly shorter in Gαi2 (-/-)-deficient mice than other groups. We next performed similar experiments in WT mice using the selective GIRK blocker tertiapinQ (Drici et al. 2000) or the muscaranic receptor antagonist atropine and reassessed HR dynamics. Our results suggest that both tertiapinQ and atropine have effects similar to global deletion of Gαi2 with respect to HRV. Additionally tertiapinQ pretreatment of WT (and Giα1/3) mice attenuates carbachol-induced bradycardia.