This study applies a minimally invasive and multi-system measurement approach (using salivary analytes) to examine associations between the psychobiology of the stress response and affective behavior in toddlers. Eighty-seven 2-year-olds (48 females) participated in laboratory tasks designed to elicit emotions and behavior ranging from pleasure/approach to fear/withdrawal. Saliva samples were collected pretask and immediately posttask, and assayed for markers of sympathetic nervous system (alpha-amylase or sAA) and hypothalamic-pituitary-adrenal axis (cortisol) activity. Individual differences in sAA were positively associated with approach behavior and positive affect; whereas, cortisol was positively associated with negative affect and withdrawal behavior. The findings suggest that individual differences in sAA may covary specifically with positive affect and approach behaviors or the predominant emotional state across a series of tasks. The results are discussed with respect to advancing biosocial models of the concomitants and correlates of young children's affective behaviors.