Cardiac autonomic tone can be assessed either by estimating separately vagal and sympathetic tones or by evaluating the net effect of their interaction, the so-called sympathovagal balance (SVB). To compare the most commonly used methods in rats, telemetric recordings of the electrocardiogram were performed in normotensive WKY rats, and in groups of spontaneously hypertensive (SHR) rats that were either untreated or chronically treated with the cholinesterase inhibitor, pyridostigmine, to enhance vagal tone. Cardiac autonomic blockers were administered alone and in combination, so that heart rate (HR) could be measured (1) under resting conditions, (2) with either autonomic branch blocked, and (3) with both branches blocked (which provided intrinsic HR, iHR). SVB was assessed as the ratio of resting HR to iHR. This calculation pointed to a sympathetic predominance in untreated SHRs and even more so in WKY rats, and to a marked vagal predominance in pyridostigmine-treated SHRs. By contrast, the ratio between low and high frequency components (LF/HF) of RR interval spectra did not significantly differ between the groups. Each autonomic tone was quantified as the HR change induced by its selective blocker or as the difference between iHR and HR after blockade of its counterpart. Both pharmacological methods indicated vagal enhancement in treated SHRs, but provided opposite results in terms of vagal vs. sympathetic predominance. These data seriously question the use of the LF/HF ratio as an index of SVB, and the possibility to reliably estimate vagal and sympathetic tones separately through current pharmacological approaches in conscious rats.