This study examined relations between maternal sensitivity as observed in a free play interaction and changes in levels of the glucocorticoid hormone cortisol in response to procedures designed to elicit negative affect in 6-month old infants. The sample included 1,292 families in predominantly rural and low-income communities in Pennsylvania and North Carolina. Results indicated that infants of more sensitive mothers had lower levels of cortisol at baseline and increased cortisol reactivity and regulation in response to the emotion procedures. Maternal negativity was unrelated to infant cortisol. Findings highlight the need for further research on variation in early caregiving and the development of the stress response in young children.