Transitioning to an out-of-home child care setting has been shown to be a challenging event for young children. One of the physiological indicators of stress, cortisol secretion, has only been studied minimally in infants yet (e.g., Albers et al., 2016). In the current replication study we therefore followed 32 healthy infants (Mage = 11.59 weeks), their mothers, and their primary professional caregivers during the transition from home to the child care center. We found that on average (1) infants’ cortisol levels were significantly lower at mid-afternoon than mid-morning both at home and child care; (2) cortisol levels at home (both time-points) were significantly higher before transition than after transition; (3) the difference between cortisol levels at child care versus home showed a (non-significant) medium effect with higher levels at child care at both time-points; (4) individual cortisol patterns illustrated large variability between infants; and (5) three exploratory correlates of cortisol secretion at child care displayed a (non-significant) small to medium effect: infants who displayed a cortisol increase over the day scored lower on infant negative emotionality, and had mothers who scored lower on sensitivity and higher on separation anxiety. Larger studies are required, including multiple caregivers and various physiological measures.