Self-regulation ability is an important component of children's academic success. Physiological reactivity may relate to brain activity governing attention and behavioral regulation. Saliva samples collected from 186 preschool children (101 boys, mean age = 53 months, 34% minority) before and after a series of mildly challenging games and again 30 min following the challenges were used to assess hypothalamic–pituitary–adrenal (HPA) (cortisol) and sympathetic nervous system (SNS; alpha-amylase) activity. Behavioral regulation was measured in delay of gratification and slow-down motor activities included in the challenge task. Cortisol and alpha-amylase were related differentially to aspects of behavioral regulation. Low cortisol was related to classroom regulation difficulties. Children with greater cortisol elevations following the challenge task had poorer performance on the slow-down motor task. Children with greater increases in salivary alpha-amylase following challenge were less able to delay gratification. Results suggest that HPA axis and SNS arousal may contribute differentially to behavioral regulation ability.