Recently, heart rate spectral analysis has become recognized as a powerful tool for quantitatively evaluating autonomic nervous system activity. The purpose of this study was to analyze parasympathetic nervous activity by heart rate spectral analysis after administration of atropine and neostigmine for reversal of residual neuromuscular blockade. For our study, 36 female patients (26-37 years of age), ASA physical status (PS) I, who were scheduled for laparoscopic examination, were randomly allocated to one of the following four groups: In group A (1∶1), 9 patients received 1.0mg atropine followed 4 min later by 1.0 mg neostigmine. In group B (1∶2), 9 patients received 0.5 mg atropine followed 4 min later by 1.0 mg neostigmine. In group C (1∶2.5), 9 patients received 1.0 mg atropine followed 4 min later by 2.5 mg neostigmine. In group D (1∶2 mix), 9 patients received a mixed solution of atropine 0.5 mg and neostigmine 1.0mg. After finishing the laparoscopic examination, additional anesthesia was maintained with 70% nitrous oxide, 30% oxygen, and 0.5% isoflurane. The control data were obtained 10 min after finishing the laparoscopic examination. After that, the data on atropine were obtained between 2 and 4min after administration of atropine, and the data on neostigmine were obtained between 5 and 7 min after administration of neostigmine. We selected power spectral density of the high-frequency component (HF-p) in heart rate spectral analysis as an index to assess parasympathetic activity. In groups A, B, and C, the HF-p decreased after administration of atropine. In groups B and C, the HF-p increased after administration of neostigmine as compared to the control. In group A, the HF-p increased after neostigmine but did not differ from the control. The difference between groups D and B was not statistically significant. From the results of this study, we concluded that the muscarinic effect of neostigmine could not be sufficiently blocked by atropine at 1/2 dosages of neostigmine, but could be sufficiently blocked by atropine at equivalent dosages of neostigmine, under light isoflurane anesthesia.