Increased deposition of hyaluronan (HA) is part of the early response to fibrogenic stimulus in the lung exposed to bleomycin injury and has been associated with increased lung water in adult animals. Early respiratory distress syndrome (RDS) in premature infants is characterized by increased lung water, and late sequelae include fibrosis or bronchopulmonary dysplasia. We hypothesized that increased MA in the alveolar interstitium would be associated with increasingly severe RDS in prematurely delivered monkeys and that modes of therapy that affect severity of disease such as treatment with high-frequency oscillatory ventilation or exogenous surfactant would decrease this response. Thirty-four Macaca nemestrina monkeys were delivered at 134 ± 1 d (term = 168 d) and randomized to high-frequency oscillatory ventilation or conventional mechanical ventilation from birth. Sixteen of these animals received surfactant. At 6 h of age, the right lower lung was frozen in situ during inflation to 30 cm H2O (approximately 2940 Pa) and then dehydrated and processed for microscopy. The presence and severity of RDS were evaluated by clinical and morphologic criteria. HA concentrations in lung extracts increased with progressively severe RDS (p = 0.0003). Treatment with high-frequency oscillatory ventilation decreased the lung injury score (1.69 ± 0.7 compared with 2.5 ± 0.9, p = 0.05), but changes in lung HA concentration did not reach significance (37.9 ± 22.7 compared with 44.8 ± 22.6). Surfactant treatment decreased lung HA concentration (29.6 ± 19.0 μg/wet lung) compared with non-surfactant-treated animals (54.7 ± 20.2 μg/g wet lung, p = 0.0009). Two fetal animals (144 and 163 d gestation) and seven additional premature animals ventilated for up to 96 h were compared with the animals killed at 6 h. HA concentrations increased with length of mechanical ventilation and severity of illness in these animals. HA was localized in freeze-dried lung sections using a biotinylated probe. Lung sections were blindly scored for the distribution of HA staining, and these scores were positively correlated with HA concentration measurements (r = 0.75, p < 0.0001). The quantity of HA in alveolar microvasculature correlated with severity of RDS (r = 0.68, p = 0.0004). We conclude that 1) HA concentration in RDS lungs of prematurely delivered infant monkeys is increased relative to normal lungs at 6 h, 2) increased HA is localized predominantly to the perivascular space of lung vasculature, and 3) this response is decreased by surfactant treatment.