A 3.6-kb EcoRI fragment containing the ntrA gene of Agrobacterium tumefaciens was cloned by using the homologous ntrA gene of Rhizobium meliloti as a probe. Construction of an ntrA mutant of A. tumefaciens by site-directed insertional mutagenesis demonstrated the requirement of the ntrA gene for nitrate utilization and C4-dicarboxylate transport but not for vir gene expression or tumorigenesis.