The purpose of this study is to describe the relationship between neonatal regulatory activities and emotional expression at 3 months. Subjects included 82 infants: 31 healthy term, 23 healthy preterm, and 28 sick preterm. All preterms were <34 weeks gestation and <1750g at birth; sick preterms had one or more of the following: respiratory illness; sepsis, NEC, or hyperbilirubinemia. The Neonatal Behavioral Assessment Scale (NBAS) was administered to the neonates and videotaped while simultaneous heart rate was collected. Mean heart rate (HR) and Respiratory Sinus Arrythmia (RSA) were calculated during alert conditions of the NBAS as coded from the videotape. Scores from NBAS items were averaged together to generate Behavioral, Motor; State, Autonomic, General regulatory, Overall regulatory and Reactivity scores. At three months, the Garcia-Coll procedure for temperament assessment consisting of 11 episodes of challenging events was presented to the infant and videotaped while simultaneous heart rate was collected. The videotape was later coded for emotional expression. HR, RSA, and the percentage of negative and positive affect were calculated. All neonatal regulation scores as well as Reactivity were associated with less negative affect (range r=.-34 to r=-56, P<.05 in all cases). The Overall regulatory score and the Behavioral, Motor, State, and General scores were associated with more positive affect at 3 months (range r=.23 tor.33 P.<05 in all cases). The Overall regulatory score and the Behavioral, Autonomic, and General scores were associated with higher RSA at 3 months (range r=.23 to r=.28,P<.05 in all cases). Finally, infants with higher RSA during NBAS events also had higher RSA during 3 month events (range r=.29 to r=.36, P<.05 in all cases). Partial correlations were calculated to extract the effects of prematurity. The association between all neonatal regulation scores and less negative affect at 3 months was retained (range r=.25 to r=.43, P<.05), but only Motor and Overall regulatory scores were significantly related to positive affect at 3 months (r=.23 in both cases,P<.05). In sum, neonatal regulation was related to emotional expression and physiological response 2 months later, suggesting that antecedents of emotional regulation may be observed during the neonatal period. Prematurity mediates the relationship with 3 months positive but not negative affective scores.