The objectives of this study were to (1) develop a behavioral assessment tool for the measurement of pain in the preterm and full-term neonate; (2) establish the construct and concurrent validity, interrater reliability, and internal consistency of the tool; and (3) examine the relationship between the pain scores and infant characteristics. Thirty-eight infants contributed to the 90 procedures videotaped for the study. The Neonatal Infant Pain Scale (NIPS) was used to score behavioral responses before, during, and after each intrusive procedure. The significant difference in NIPS scores over time indicates that the scale provides a measurement of intensity of infant responses to intrusive procedures. Concurrent validity was established by correlations, ranging from .53 to .84, between NIPS scores at each minute of observation and scores on the Visual Analogue Scale. Interrater reliability was high: Pearson correlations ranged from .92 to .97 across successive minutes of observation. The six component scores of the NIPS had high internal consistency: Cronbach's alphas were .95, .87, and .88 for before, during, and after the procedures, respectively. Although gestational age and five-minute Apgars were positively associated with NIPS scores over time, there was no association between these factors and responsiveness to pain, as measured by change in NIPS scores from before to during the procedure. Results are discussed in terms of the use of the NIPS in clinical trials and its clinical application in a neonatal intensive care unit.