Pseudomonas aeruginosa causes chronic pulmonary infections, which can persist for decades, in patients with cystic fibrosis (CF). Current evidence suggests that the glyoxylate pathway is an important metabolic pathway for P. aeruginosa growing within the CF lung. In this study, we identified glcB, which encodes for the second key enzyme of the glyoxylate pathway, malate synthase, as a requirement for virulence of P. aeruginosa on alfalfa seedlings. While expression of glcB in PAO1, an acute isolate of P. aeruginosa, responds to some carbon sources that use the glyoxylate pathway, expression of glcB in FRD1, a CF isolate, is constitutively upregulated. Malate synthase activity is moderately affected by glcB expression and is nearly constitutive in both backgrounds, with slightly higher activity in FRD1 than in PAO1. In addition, RpoN negatively regulates glcB in PAO1 but not in FRD1. In summary, the genes encoding for the glyoxylatespecific enzymes appear to be coordinately regulated, even though they are not located within the same operon on the P. aeruginosa genome. Furthermore, both genes encoding for the glyoxylate enzymes can become deregulated during adaptation of the bacterium to the CF lung.