Acute pain in infants is not assessed or managed optimally. The objectives of the study were (a) to adapt a behavioral pain assessment measure (Children's Hospital of Eastern Ontario Pain Scale, CHEOPS) for use with infants, and (b) to establish the reliability and validity of the measure in a study of infants undergoing immunization. Ninety-six healthy 4- to 6-month-old infants were randomized to receive either the local anesthetic cream Eutectic Mixture of Local Anesthetics (EMLA) (N = 49), or a placebo (N = 47) prior to immunization. The infant's behavioral response was videotaped immediately before and following the immunization. Postprocedural pain scores were assessed from the videotape and were significantly lower in infants who received EMLA (P = 0.01). Pain scores were also significantly correlated with visual analogue scale (VAS) scores assessed during vaccination. Five independent raters also independently rated ten infants to determine interrater reliability. Agreement between raters' scores was high (intraclass correlation coefficient, 0.95). Results from this study suggest that this measure has beginning construct and concurrent validity and interrater reliability when used in a research study. Further testing of the measure in the clinical setting is required.