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The objective of this study is to determine whether use of lidocaine–prilocaine 5% cream (EMLA®) and oral glucose decreases pain associated with diphteria–pertussis–tetanus (DPT) immunization in 3-month-old infants. Design: randomized, double-blind, controlled trial in outpatient paediatric practice in northern Sweden. EMLA or placebo was applied to the infant's lateral region of the right thigh and covered with an occlusive dressing 1 h before the immunization. In addition, 1 ml of glucose (300 mg/ml) or placebo (water) was instilled on the baby's tongue within 2 min before the DPT-injection. Forty-five infants received EMLA and glucose and 45 infants placebo cream and water. ECG was recorded and stored in a computer and the procedure was videotaped. The parents and the nurse assessed the infants' pain on a visual analogue scale (VAS) after the immunization. Heart rate and heart rate variability pre- and post-injection were calculated. From the videotapes, the modified behavioural pain scale (MBPS) was used to assess pain scores during baseline and after immunization. The latency of the first cry and total crying time were measured. The parents and the nurse scored the infants' pain on the VAS significantly lower in the treatment group than in the placebo group. The infants' responses to the immunization measured as the difference in MBPS scores pre- and post-injection were significantly lower in the EMLA–glucose group compared with the placebo group. More infants cried after the immunization in the placebo group compared with the EMLA–glucose group and the latency of the first cry after the injection was shorter in the placebo group. A biphasic transient heart rate response with a marked deceleration followed by a subsequent acceleration was seen more frequently in the placebo group compared to the EMLA–glucose group. EMLA and glucose alleviate immunization pain in 3-month-old infants.