Research Findings: Measuring toddlers' cortisol levels both in childcare and at home and their relation to child- and childcare-related factors may help to identify stress-inducing childcare practices and children who are more vulnerable to stress in childcare. Accordingly, toddlers' (n = 320, 51.2% female, mean age = 26.8 months) cortisol levels in childcare and at home and their relation to childcare quality and child- and family-related factors were investigated using linear mixed model analyses. Mid-morning to mid-afternoon cortisol levels increased in childcare and decreased at home. Younger children had higher overall cortisol levels. Children attending groups with a higher caregiver-child ratio had slightly higher cortisol levels in childcare and slightly lower cortisol levels at home. Toddlers attending disorganized groups were considerably more stressed in childcare. Practice or Policy: The present study underlines the importance of sufficient caregiver availability and stable routines in toddler classrooms.