The effects of breathing greater than 95% oxygen from birth for 48 h of life on surfactant phosphatidylcholine synthesis and secretion, as well as uptake and stability of exogenous phosphatidylcholine were studied using rabbit lung tissue slices. Lung slices from animals breathing greater than 95% oxygen for 48 h exhibited a decreased rate of [14C]phosphatidylcholine release (30%) in comparison to lung slices from air-exposed controls. In vitro incorporation of [14C]choline into phosphatidylcholine was decreased by a similar amount in lung slices from pups exposed to greater than 95% oxygen. Uptake of exogenous [14C]phosphatidylcholine by lung slices from hyperoxic-exposed and control groups was not different, and the stability of extracellular phosphatidylcholine was likewise unaffected by hyperoxia. Turnover of labelled phosphatidylcholine taken up by tissue slices from medium was apparently decreased in association with hyperoxic exposure. These results are consistent with multiple sites of effect of hyperoxia on the pulmonary surfactant system in the newborn. These effects of hyperoxia on the lung surfactant system occur at a time of critical adaption to extrauterine life, and thus may have major consequences on lung function and ultimate survival of the premature infant with respiratory distress syndrome.