Atrial fibrillation (AF) is the most common arrhythmia seen in general practice. Muscarinic ACh receptors (M2R, M3R) are involved in vagally induced AF. M2R and M3R activate the heterotrimeric G proteins, G(i) and G(q), respectively, by promoting GTP binding, and these in turn activate distinct K(+) channels. Signaling is terminated by GTP hydrolysis, a process accelerated by regulator of G protein signaling (RGS) proteins. RGS2 is selective for G(q) and thus may regulate atrial M3R signaling. We hypothesized that knockout of RGS2 (RGS2(-/-)) would render the atria more susceptible to electrically induced AF. One-month-old male RGS2(-/-) and C57BL/6 wild-type (WT) mice were instrumented for intracardiac electrophysiology. Atrial effective refractory periods (AERPs) were also determined in the absence and presence of carbachol, atropine, and/or the selective M3R antagonist darifenacin. Susceptibility to electrically induced AF used burst pacing and programmed electrical stimulation with one extrastimulus. Real-time RT-PCR measured atrial and ventricular content of RGS2, RGS4, M2R, M3R, and M4R mRNA. AERP was lower in RGS2(-/-) compared with WT mice in both the high right atrium (HRA) (30 +/- 1 vs. 34 +/- 1 ms, P < 0.05) and mid right atrium (MRA) (21 +/- 1 vs. 24 +/- 1 ms, P < 0.05). Darifenacin eliminated this difference (HRA: 37 +/- 2 vs. 39 +/- 2 ms, and MRA: 30 +/- 2 vs. 30 +/- 1, P > 0.4). RGS2(-/-) were more susceptible than WT mice to atrial tachycardia/fibrillation (AT/F) induction (11/22 vs. 1/25, respectively, P < 0.05). Muscarinic receptor expression did not differ between strains, whereas M2R expression was 70-fold higher than M3R (P < 0.01). These results suggest that RGS2 is an important cholinergic regulator in the atrium and that RGS2(-/-) mice have enhanced susceptibility to AT/F via enhanced M3 muscarinic receptor activity.