Plasma and bronchoalveolar lavage (BAL) biomarkers related to the pathogenesis of acute lung injury (ALI) have previously been associated with poorer clinical outcomes and increased disease severity among patients with ALI. Whether these biomarkers have predictive value in a less severely ill population that excludes septic patients with high APACHE II scores is currently unknown. We tested the association of plasma and BAL biomarkers with physiological markers of ALI severity or clinically relevant outcomes in a secondary analysis of a clinical trial of activated protein C for the treatment of ALI. Plasma plasminogen activator inhibitor-1 (PAI-1) and mini-BAL protein were both significantly associated with increased oxygenation index (P = 0.02 and 0.01, respectively), whereas there was a trend toward an association between IL-6 and oxygenation index (P = 0.057). High plasma IL-6, thrombomodulin, and mini-BAL protein were all significantly associated with fewer ventilator-free days (VFDs) (P = 0.01, 0.01, and 0.05, respectively); no markers were associated with mortality, but we hypothesized that this was due to the small size of our cohort and the low death rate. To confirm these associations in a larger sample, we identified a restricted cohort of patients from the ARDS Network ALVEOLI study with similar baseline characteristics. We retested the associations of the significant biomarkers with markers of severity and clinical outcomes and studied IL-8 as an additional biomarker given its important predictive value in prior studies. In this restricted cohort, IL-6 was significantly associated with oxygenation index (P = 0.02). Both IL-6 and IL-8 were associated with decreased VFDs and increased 28-day mortality. Future studies should be focused on examining larger numbers of patients with less severe ALI to further test the relative predictive value of plasma and mini-BAL biomarkers for clinically relevant outcomes, including VFDs and mortality, and for their prospective utility in risk stratification for future clinical trials.