Background: Short-acting opioids like remifentanil are suspected of an increased risk for tolerance, withdrawal and opioid-induced hyperalgesia (OIH). These potential adverse effects have never been investigated in neonates. Objectives: To compare remifentanil and fentanyl concerning the incidence of tolerance, withdrawal and OIH. Methods: 23 mechanically ventilated infants received up to 96 h either a remifentanil- or fentanyl-based analgesia and sedation regimen with low-dose midazolam. We compared the required opioid doses and the number of opioid dose adjustments. Following extubation, withdrawal symptoms were assessed by a modification of the Finnegan score. OIH was evaluated by the CHIPPS scale and by testing the threshold of the flexion withdrawal reflex with calibrated von Frey filaments. Results: Remifentanil had to be increased by 24% and fentanyl by 47% to keep the infants adequately sedated during mechanical ventilation. Following extubation, infants revealed no pronounced opioid withdrawal and low average Finnegan scores in both groups. Only 1 infant of the fentanyl group and 1 infant of the remifentanil group required methadone for treatment of withdrawal symptoms. Infants also revealed no signs of OIH and low CHIPPS scores in both groups. The median threshold of the flexion withdrawal reflex was 4.5 g (IQR = 2.3) in the fentanyl group and 2.7 g (IQR = 3.3) in the remifentanil group (p = 0.312), which is within the physiologic range of healthy infants. Conclusions: Remifentanil does not seem to be associated with an increased risk for tolerance, withdrawal or OIH.