Prenatal psychosocial exposures can significantly affect infant health and development. Infants with higher temperamental negativity are theorized to be more susceptible to environmental exposures. We evaluated the interaction of prenatal maternal exposures and infant temperamental negativity to predict infant cortisol response to mildly challenging mother-infant interaction tasks. Participants included 322 Mexican American mother-infant dyads (mother age 18–42; 82% Spanish-speaking; modal family income $10,000–$15,000). Mothers reported depressive symptoms and social support prenatally and infant temperamental negativity at 6 weeks postpartum. Salivary cortisol was collected from infants before and after mother-infant interaction tasks at 12 weeks. Higher prenatal maternal depressive symptoms and lower social support predicted higher cortisol among infants with higher temperamental negativity. Higher infant temperamental negativity predicted an increase in maternal distress and a decrease in social support from prenatal to 12 weeks postpartum. Interactive influences of maternal social-contextual factors and infant temperament may influence the development of infant neurobiological regulation and promote or strain maternal and infant adaptation over time. © 2015 Wiley Periodicals, Inc. Dev Psychobiol 57: 961–973, 2015.