In this investigation we found that ascorbate and urate levels in the bronchoalveolar lavage (BAL) of premature infants with respiratory distress syndrome (RDS) was significantly increased after administration of surfactant(Exosurf or Survanta). RDS is a major cause of morbidity and mortality in preterm infants. An estimated 50% of all neonatal deaths result from RDS or its complications. Although many of the clinical complications of RDS have been linked to oxidative stress there have been few biochemically oriented studies of this problem. A major obstacle has been the availability of microanalytical methods with sufficient sensitivity. Our long term goal is to study the interrelationships between RDS, oxygen toxicity and the antioxidant status of preterm infants receiving surfactant. Ascorbate and urate are major water soluble antioxidants that inhibit free radical pathology. In this pilot study, BAL samples were obtained from seven intubated preterm infants and a preservative (dithioerythritol) and internal standards immediately added. A unique sodium dodecylsulfate-micellar electrokinetic chromatography (MEKC) method was developed to assay BAL levels of water soluble antioxidants. The method utilized theophylline as an internal standard for urate and isoascorbate as an internal standard for ascorbate. Pre- and post surfactant urate levels (means±SEM) were 16.4±10.6 μM and 64.4±19.6 μM, whereas ascorbate levels were 2.5±2.5 μM and 12.8±8.3 μM. This research was partially funded by a grant from Burroughs Wellcome Co.