It is generally accepted that cardiac sympathetic tone dominates the control of heart rate (HR) in mice. However, we have recently challenged this notion given that HR in the mouse is responsive to ambient temperature (T_a) and that the housing T_a is typically 21-23°C, well below the thermoneutral zone (~30°C ) of this species. To specifically test the hypothesis that cardiac sympathetic tone is the primary mediator of HR control in the mouse, we first examined the metabolic and cardiovascular responses to rapid changes Ta to demonstrate the sensitivity of the mouse cardiovascular system to T_a. We then determined HR in: 1) mice deficient in cardiac sympathetic tone (-less mice), 2) mice deficient in cardiac vagal tone (M₂R -/- mice), and 3) littermate controls. At a T_a of 30°C, the HR of -less mice was identical to that of wild type mice (351 ± 11, 363 ± 10 bpm, respectively). However, the HR of M₂R -/- mice was significantly greater (416 ± 7 bpm), demonstrating that vagal tone predominates over HR control at this T_a. When these mice were calorically restricted (CR) to 70% of normal intake, HR fell equally in wild type mice, -less, and M₂R -/- (HR = 73 ± 9, 76 ± 3, 73 ± 7 bpm, respectively), suggesting that the fall in intrinsic HR governs bradycardia of CR. Only when the T_a was relatively cool, at 23°C, did -less mice exhibit a HR (442 ± 14 bpm) that was not different from that of littermate controls (604 ± 10 bpm) and M₂R -/- (602 ± 5 bpm). These experiments conclusively demonstrate that in the absence of cold stress, regulation of vagal tone and modulation of intrinsic rate are important determinants of HR control in the mouse.