The purpose of this study was to characterize changes in the endogenous surfactant system in adult sheep rendered septic via cecal ligation and perforation (CLP). Forty-eight hours after CLP, septic animals had significant increases in mean pulmonary artery pressure (PAP) (p < 0.01), cardiac index (CI) (p < 0.01), and arterial lactate (p < 0.05) values compared with their respective baseline values, while the same measurements in a sham-operated control group did not change significantly. The changes in the septic group were associated with significantly lower PaO2 (p < 0.05) and alveolar to arterial (A-a) oxygen gradient values (p < 0.01) at 48 h compared with baseline measurements. No changes in oxygenation occurred within the sham-operated group. Surfactant phospholipid (PL) composition and surface activity measurements of isolated alveolar surfactant aggregate forms were similar for the two groups at 48 h. However, the ratio of poorly functioning small aggregate forms (SA) to superior functioning large aggregates (LA) was significantly increased in the septic versus the sham-operated animals (p < 0.01). This was associated with significantly decreased surfactant protein A (SP-A), B (SP-B), and C (SP-C) levels in septic versus sham-operated animals (p < 0.05). We conclude that sepsis-associated lung injury resulted in altered alveolar surfactant aggregate forms. These changes, together with altered surfactant protein levels, may represent a very sensitive marker of acute lung injury in high-risk patients. Furthermore, these findings suggest that exogenous surfactant given at an early stage of lung injury may mitigate progressive lung dysfunction.