A role for polyamines during growth of Agrobacterium tumefaciens was investigated. alpha-Difluoromethylornithine (DFMO, 1 mM), cyclohexylamine (CHA, 10 mM) or alpha-difluoromethyllysine (DFML, 1 mM), inhibitors of putrescine (PUT), spermidine (SPD), and cadaverine (CAD) biosynthesis, respectively, reduced growth on minimal medium by up to 63%. Growth inhibition by DFMO was reversed by 1 mM PUT, but SPD and CAD did not reverse the effects of CHA or DFML, respectively. Alpha-difluoromethylarginine (1-10 mM), an inhibitor of arginine decarboxylase, did not inhibit growth. PUT and SPD were the predominant polyamines present in control A. tumefaciens cells at late log phase, and DFMO reduced their levels by greater than 75%. DFML reduced polyamine levels to a lesser extent, and CHA had no effect. In extracts from control cells, significant ornithine decarboxylase (ODC) activity was detected, while no specific arginine or lysine decarboxylase activity was evident. In vitro, ODC activity was inhibited 98% by 0.75 mM DFMO and 33% by 0.75 mM DFML. Exposure of cells to 1 mM DMFO for 1 h reduced extractable ODC activity by greater than 90%. These data suggest that polyamines are important for growth of A. tumefaciens and that ODC is the primary enzyme responsible for PUT biosynthesis in these cells.