Background: The association of plasma interleukin-8 (IL-8), or IL-8 genetic variants, with pediatric acute respiratory distress syndrome (PARDS) in children with acute respiratory failure at risk for PARDS has not been examined. The purpose of this study was to examine the association of early and sequential measurement of plasma IL-8 and/or its genetic variants with development of PARDS and other clinical outcomes in mechanically ventilated children with acute respiratory failure. Methods: This was a prospective cohort study of children 2 weeks to 17 years of age with acute airways and/or parenchymal lung disease done in 22 pediatric intensive care units participating in the multi-center clinical trial, Randomized Evaluation of Sedation Titration for Respiratory Failure (RESTORE). Plasma IL-8 levels were measured within 24 h of consent and 24 and 48 h later. DNA was purified from whole blood, and IL-8 single nucleotide polymorphisms, rs4073, rs2227306, and rs2227307, were genotyped. Results: Five hundred forty-nine patients were enrolled; 480 had blood sampling. Plasma IL-8 levels ranged widely from 4 to 7373 pg/mL. Highest IL-8 levels were observed on the day of intubation with subsequent tapering. Levels of IL-8 varied significantly across primary diagnoses with the highest levels occurring in patients with sepsis and the lowest levels in those with asthma. Plasma IL-8 was strongly correlated with oxygenation defect and severity of illness. IL-8 was consistently higher in PARDS patients compared to those without PARDS; levels were 4-12 fold higher in non-survivors compared to survivors. On multivariable analysis, IL-8 was independently associated with death, duration of mechanical ventilation, and PICU length of stay on all days measured, but was not associated with PARDS development. There was no association between the IL-8 single nucleotide polymorphisms, rs4073, rs2227306, and rs2227307, and PARDS development or plasma IL-8 level. Conclusions: When measured sequentially, plasma IL-8 was robustly associated with multiple, relevant clinical outcomes including mortality, but was not associated with PARDS development. The wide range of plasma IL-8 levels exhibited in this cohort suggests that further study into the heterogeneity of this patient population and its impact on individual responses to PARDS treatment is warranted.