PURPOSE: Stress related to dental treatment can be associated with negative outcomes. Heart rate variability (HRV) is an objective measurement of autonomic nervous system activity. Therefore, HRV was used to identify autonomic nervous system reactions during mesiodens extraction under general anesthesia in children. MATERIALS AND METHODS: Electrocardiography was performed with customized software during treatment. HRV parameters were analyzed according to time and frequency domains during each dental procedure (local anesthesia, incision, flap, bone removal, extraction of mesiodens, and suturing). The relations of HRV parameters to age also were determined. RESULTS: Total autonomic nervous system activity decreased markedly after local anesthesia injection. Depending on the responses of sympathetic nerve activity, patients were categorized in a stress group and a nonstress group. The ratio of low-frequency power (LF) to high-frequency power (HF), an indication of sympathetic and parasympathetic balance, increased in the stress group after incision and flap formation. Conversely, the LF/HF ratio decreased during treatment in the nonstress group. However, HR, widely used to evaluate stress responses, did not change statistically during mesiodens extraction in either group. HRV parameters did not differ statistically according to age. CONCLUSIONS: The internal stress related to mesiodens extraction can be evaluated more objectively with HRV parameters than with conventional methods. Sympathetic nerve activity in the stress group differed from that in the nonstress group during the treatment procedures.