Pseudomonas aeruginosa often establishes a chronic infection in the airways of patients with cystic fibrosis (CF). l-Lactate is the most abundant carbon source in the CF sputum, and l-lactate utilization may be important for P. aeruginosa to survive in the lungs of CF patients. In this study, the key enzymes involved in l-lactate utilization by P. aeruginosa PAO1 were characterized using the synthetic CF sputum medium (SCFM). A highly conserved membrane-bound NAD-independent l-lactate dehydrogenase (l-iLDH) encoded by lldD (PA4771) and a novel flavin-containing membrane-bound l-iLDH encoded by lldA (PA2382) were both found to contribute to l-lactate utilization by P. aeruginosa PAO1. In addition, an lldD and lldA double mutant was incapable of growing in a medium containing l-lactate as the sole carbon source. This study clarifies the mechanism and importance of l-lactate catabolism, and demonstrates the first Pseudomonas spp. expressing two l-lactate-oxidizing enzymes.