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Teacher–child relationships were examined as predictors of cortisol change in preschool children. Saliva for assays was collected from one hundred and ninety-one 4-year-olds (101 boys) in the mornings and afternoons on 2 days at child care, and before and after a series of challenging tasks and a teacher–child interaction session outside the classroom. Parents reported on children’s temperament, teachers and children reported on teacher–child relationship quality, and observers rated group-level teacher insensitivity. Teacher-reported relationship conflict predicted cortisol increases during teacher–child interaction and teacher-reported overdependence predicted cortisol increases from morning to afternoon, even after controlling for individual teacher, child, and classroom characteristics. The findings extend earlier work by suggesting that cortisol change across the child-care day is influenced by teacher–child relationship characteristics.