Hospitalized infants undergo repeated invasive procedures. It is unknown whether cumulative experiences with pain lead to anticipatory pain behaviors and hyperalgesia. To determine whether newborns who are born to mothers with diabetes and undergo repeated pain learn to anticipate pain and exhibit more pain during a painful procedure than normal infants. Prospective cohort study of 21 full-term newborns born to mothers with diabetes and 21 born to mothers with uneventful pregnancies, at a university teaching hospital between August 1999 and October 2000. Infants of diabetic mothers underwent repeated heel lances in the first 24 to 36 hours of life for monitoring of blood glucose concentrations. Pain responses of all infants undergoing a venipuncture on the dorsum of the hand to obtain blood for the newborn screening test after the first day of life were compared. In addition, from September through November 2001, 12 infants of diabetic mothers and 12 normal infants were compared for pain reactions to intramuscular vitamin K injection after birth. Percentages of time observed grimacing and crying and visual analog scale (VAS) scores. Raters were blinded to exposure group. Median baseline scores for grimacing, crying time, and VAS did not differ significantly between groups (P =.27, P =.32, and P =.32, respectively). Median scores (interquartile range) for grimacing during skin cleansing were higher in infants of diabetic mothers (22.2% [77.5%] vs 0% [15%]; P =.03). The VAS scores for both groups were zero, but the distribution of the scores was significantly different (86% of normal infants vs 52% of infants of diabetic mothers had scores of zero) (P =.04). During venipuncture, infants of diabetic mothers had higher median scores for grimacing (81.7% [32.5%] vs 40% [73.4%]; P =.01), VAS (69% [27.5%] vs 5% [60.5%]; P =.002), and crying (40.2% [77%] vs 0% [54.8%]; P =.03) compared with normal infants. There were no differences between groups on any pain measure in response to intramuscular injection. Newborns who had diabetic mothers and were exposed to repeated heel lances in the first 24 to 36 hours of life learned to anticipate pain and exhibited more intense pain responses during venipuncture than normal infants.