The characteristics of14C-proline uptake by cells of Agrobacterium tumefaciens are described. The results obtained were different from those reported for other bacteria (e.g. Escherichia coli). In addition, significant differences were observed with cells of the non-tumorogenic strain, IIBNV6, as compared to the tumorogenic strain, IIBV7K.Uptake of14C-proline by both strains of cells did not show the specificity known to exist for this amino acid in other microorganisms. A host of unrelated amino acids inhibited proline uptake. Moreover, the inhibitory effects of some amino acids were different in the two strains. Although glutamate and cysteine had no effect on14C-proline uptake in IIBNV6cells, they showed strong inhibition in IIBV7K cells. In addition, cells of the latter strain metabolized proline to a much greater extent than IIBNV6cells.In growing IIBV7K cells, glycine and glutamate suppressed14C-proline uptake and its incorporation into protein. They were also able to displace accumulated14C-proline from the cells. Glycine and leucine, which inhibited proline uptake in IIBNV6cells, showed a similar effect on the displacement of accumulated14C-proline in these cells.Hydroxylamine and Δ′-pyrroline carboxylate suppressed14C-proline uptake and its incorporation into protein in IIBNV6ceils. In IIBV7K cells, on the other hand, Δ′-pyrroline carboxylate had no effect on14C-proline uptake. The incorporation of the label into protein was only slightly reduced. Hydroxylamine in these cells showed a lesser degree of uptake inhibition but a stronger suppression of label incorporation into protein.