Heart rate (HR) and heart rate variability (HRV) are risk markers in cardiac disease. HRV is also an index of the sympathovagal modulation of heart rate. Their relations have been rarely analyzed. We aimed to study such relations in normal adult conscious rats by using a novel bradycardic agent, a sinus node inhibitor, S-16257. Placebo-drug crossover designs were used while monitoring HR with telemetry and analyzing HRV in both time and frequency domains. S-16257 (2 mg/kg; n = 10) decreased HR by 29% and markedly increased HRV in parallel. By using various combinations of S-16257, atropine (2 mg/kg), and propranolol (4 mg/kg), a positive relation was shown between RR interval and various indexes of HRV: the slower the HR, the greater the HRV. Nevertheless, there is one exception to this correlation. When S-16257 was associated with both atropine and propranolol, the deep bradycardia was accompanied by a reduction of HRV, which indicates that the physiologic negative correlation between HR and HRV is not an intrinsic property of the pacemaker but is highly dependent on the two components of the autonomic system.