The requirement for the mobA gene in key assimilatory and respiratory nitrogen metabolism of Pseudomonas aeruginosa PAO1 was investigated by mutational analysis of PA3030 (mobA; MoCo guanylating enzyme), PA1779 (nasA; assimilatory nitrate reductase), and PA3875 (narG; respiratory nitrate reductase). The mobA mutant was deficient in both assimilatory and respiratory nitrate reductase activities, whereas xanthine dehydrogenase activity remained unaffected. Thus, P. aeruginosa requires both the molybdopterin (MPT) and molybdopterin guanine dinucleotide (MGD) forms of the molybdenum cofactor for a complete spectrum of nitrogen metabolism, and one form cannot substitute for the other. Regulation studies using a Phi(PA3030-lacZGm) reporter strain suggest that expression of mobA is not influenced by the type of nitrogen source or by anaerobiosis, whereas assimilatory nitrate reductase activity was detected only in the presence of nitrate.