This study was performed to investigate the effect of fentanyl alone on heart rate variability (HRV) during mechanical ventilation using power spectral analysis. Arterial baroreceptor reflex was also tested with pharmacological manipulation to assess the contribution of vagal baroreceptor reflex modulation of HRV during fentanyl anesthesia. Ten patients participated in this study. Electrocardiograms and arterial pressure were recorded prior to and during fentanyl (10 μg·kg−1) and vecuronium (0.2 mg·kg−1) anesthesia, with respiratory rate and tidal volume controlled ventilation. R-R intervals were analyzed by fast Fourier transformation, and changes in low-frequency (LF) and high-frequency (HF) power were compared. Arterial baroreceptor reflex regulation was also tested with administration of nitroglycerin (250 μg) or phenylephrine (250 μg). HF power was significantly reduced during anesthesia from 3.20±2.93 to 0.46±0.48 ms2·Hz−1·103 (mean±SD,P<0.05). However, LF power did not change despite increases in plasma catecholamine concentrations. The response to phenylephrine was reduced during fentanyl anesthesia from 16.6±5.7 to 9.5±5.4 ms·mmHg−1 (P<0.05), whereas the response to nitroglycerin was not affected. Our data indicate that fentanyl modulates the respiratory frequency fluctuation of HRV. This is partly caused by the effects of fentanyl on arterial baroreflex sensitivity.