The mammalian respiratory system is immature at birth. In mice, this immaturity is characterized by a fragile and highly variable breathing pattern interspersed with apneas and sigh-like breaths during postnatal days 1-3 (P1-3). Around P3-P4, the respiratory system undergoes a step in maturity, after which breathing pattern is less variable and frequency increases. The neural mechanisms underlying this maturity step are unclear. Evidence from in vitro studies suggests that two distinct medullary neuronal clusters, the opiate-sensitive preBötzinger Complex (preBötC) and the opiate-insensitive Retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG)1,2,3, play a critical role in generating respiratory rhythm but little is known of their interaction during development and early post natal life when the respiratory system is fragile. To pharmacologically tease apart the function of these neuronal clusters during early postnatal maturation of breathing and to investigate the long term effects of opiates on breathing, neonatal mice were exposed to the µ-opioid receptor agonist fentanyl (0.08mg/kg i.p daily.), or saline as a control, from P1-5 (n=16) or P9-13 (n=16). All procedures were carried out in accordance with current UK legislation. Mice were continuously monitored post-injection and breathing recorded by closed plethysmography at regular intervals from 5 minutes to 2 hours post-injection. Fentanyl had a modest effect on breathing at all postnatal days by increasing variability, decreasing frequency (250±20 vs 150±30 breaths per minute) and increasing the number of apnoeas compared to saline-exposed mice (2±1 vs 5±2 per minute respectively). At 6 weeks of age, all saline and fentanyl exposed mice were exposed to a further dose of fentanyl (0.04 – 1.0mg/kg i.p.) and monitored as above. Respiratory frequency was significantly decreased (190±10 vs120±15 breaths per minute, p<0.05 two-way anova) in all mice previously exposed to saline as neonates (P1-P5 and P9-13); however, in mice previously exposed to fentanyl as neonates (P1-P5 and P9-13) fentanyl exposure in adulthood had no effect on respiratory frequency (180±8 vs 150±10 bpm, p>0.05 two way anova). Tidal volume increased slightly in all mice post fentanyl regardless of whether they had previous exposure of fentanyl or saline. These data suggest that the respiratory system in younger animals is less susceptible to fentanyl compared to adults, and that pre-exposeure to fentanyl during early postnatal maturation results in a long term desensitization to further fentanyl insults.