Early preterm neonates in the Neonatal Intensive Care Unit (NICU) are subjected to repeated painful procedures which could sensitize their responses to pain and potentiate neuroendocrine and behavioral responses to subsequent stressors in the long-term. In this study, we used the model of the neonatal rat to test the effects of repeated pain during the first 2 weeks of life on neuroendocrine responses (CRF, ACTH and corticosterone) to stressors varying in intensity and on maternal behavior in the postnatal period. To closely mimic the type of repeated painful stimulus experienced by preterm neonates (i.e., heelstick), neonatal rats aged day 2-14 were submitted daily to having their rear heels warmed to 34 degrees C and pricked (handled and pain, HP) or not (handled, H) with a needle. For the procedure, all pups were separated from their mothers for a total period of 15 min and reunited afterwards. Unhandled (UH) pups not subjected to daily maternal separation were used as controls. On days 6 and 12, litters from the HP and H groups were videotaped for 90 min upon return with the mother and maternal behavior was analyzed. Frequency of ultrasonic vocalizations (USV) were recorded during the procedure and upon return of pups with the mother. On day 15 and 20, rat pups from all groups were exposed to a 3-min ether vapor stressor or to an openfield for 10 min. Plasma ACTH and corticosterone concentrations were determined at 0, 5, 30, 60 and 120 min after stress onset. Our results show that repeated pain did not modify body weight of the pups, however, on day 6 of life, maternal pup grooming was increased significantly (P<0.05) in the HP group compared to the H group. Frequency of USV was not changed between H and HP rats either during the separation or after reunion with the mother. Plasma ACTH and corticosterone levels under basal or stimulated conditions were not different between UH, H and HP groups. However, the UH pups showed a tendency towards higher ACTH secretion after stress compared to H and HP groups. These results suggest that repeated pain during the first 2 weeks of life in the rat does not lead to significant changes in stress responsiveness in 2-week-old pups, but we suggest that changes in mother-pup interaction (increased grooming) might act as a buffer on the cumulative effect of pain on stress responsiveness.