This study examined physiological effects of separation and reunion in a sample 3- to 6-year-old children. Using continuous ambulatory recording, changes in heart rate (HR), respiratory sinus arrhythmia (RSA), and pre-ejection period (PEP) were compared across the episodes of a separation–reunion procedure based on the strange situation. RSA decreased significantly over the course of the procedure as well as on separation from the parent and not the stranger, supporting that separation from the attachment figure elicited vagal withdrawal in young children. The absence of significant PEP effects suggest that the separation–reunion procedure, and more specifically separation from the parent, was not threatening enough to activate the sympathetic nervous system, even if children were insecure attached and inhibited with regard to strangers. Some of the variability in HR increases to reunion was explained by younger age. The findings highlight the role of the ANS as a regulatory process in the parent–child relationship. © 2007 Wiley Periodicals, Inc. Dev Psychobiol 49: 119–128, 2007.