OBJECTIVE The present survey was conducted to investigate the perceptions among nurses of neonatal pain and the associated use of pharmacologic measures (PMs) and nonpharmacologic comfort measures (CMs) in neonatal intensive care units (NICUs). Pain perception, the necessity and actual use of PMs and CMs, and their relationships were investigated and compared according to nurses' positions, educational levels, the existence of guidelines, and prior education on neonatal pain management. METHODS Participants were 141 nurses from five NICUs at university hospitals. A questionnaire was developed by researchers based on previous studies of neonatal pain management and current practices in surveyed NICUs. Five-point Likert scales were used to assess nurses' perceptions of pain, the necessity of PMs and CMs, and their actual use in 29 painful procedures. RESULTS The mean scores of perceived pain and the necessity of PMs and CMs were 3.68, 2.96, and 3.79 points, respectively. The actual use of PMs and CMs was 1.67 and 2.63 points, respectively. The perceived necessity of PMs correlated with the actual use of PMs (r = .316, p < .001), and CMs were performed (r = .390, p < .001). Keeping or reading guidelines, or receiving education on pain management resulted in a higher perception of the necessity of PMs. CONCLUSIONS Korean nurses in NICUs often underestimate the necessity of pain relief measures and use few PMs or CMs. Therefore, systematic approaches to implement guidelines, such as adaptation of guidelines for each NICU, dissemination of guideline content to all NICU staff, and regular measurements of compliance with the guidelines, are recommended.