Patients with cystic fibrosis are severely affected by an infection withPseudomonas aeruginosa,a microbe known to synthesize phospholipase C. This study was designed to determine whether that enzyme would affect the function of pulmonary surfactant phospholipids. Mucoid and nonmucoid strains ofP. aeruginosa, freshly obtained from patients with cystic fibrosis, were cultured for 12 h on agar plates. The bacteria were suspended in saline solution and then pelleted by centrifugation. The supernatant was used to dilute the surfactant preparation, calf lung surfactant extract, from 35 to 2 mg/mL. Surfactant function, before and after incubation, was examined with a capillary surfactometer, an instrument specifically developed for an evaluation of the ability of surfactant to maintain patency of a narrow glass tube, simulating a terminal conducting airway. Phospholipid hydrolysis was also evaluated biochemically by determining the total content of phospholipids in surfactant before and after incubation. In five experiments, the lipids were separated with thin-layer chromatography, and the phosphorus content was determined in the diacylphosphatidylcholine band before and after incubation for 6, 24, and 48 h. Capillary openness and phospholipid concentration decreased as enzyme concentration and time of incubation increased (p< 0.0001). Linear regression showed a significant correlation between time of capillary openness and phospholipid concentration (r= 0.957;p< 0.0001). Calf lung surfactant extract hydrolysis was catalyzed by extracts of the bacteria, particularly the nonmucoid, analogous to the catalysis observed with phospholipase C. Surfactant hydrolysis catalyzed by enzymes fromP. aeruginosamight severely affect surfactant function provided enzyme concentration is high and time of incubation is long.