The characteristics of mannopine and mannopinic acid utilization by Agrobacterium tumefaciens B6S3, Arthrobacter sp. strain MBA209, and Pseudomonas putida NA513 were studied. Strain B6S3 utilized the four mannityl opines, mannopine, mannopinic acid, agropine, and agropinic acid. It also utilized several mannityl opine analogs, which were modified in either the sugar or the amino acid moiety. It utilized mannopine more rapidly after preincubation on mannopine, mannopinic acid, or glutamine than after pregrowth on glucose, mannose, or mannitol. Strains MBA209 and NA513 utilized mannopine and mannopinic acid, but not the other two mannityl opines. They utilized few mannityl opine analogs, sometimes because of failure to utilize the products of initial cleavage of the analog. Utilization of mannopine and mannopinic acid by strain NA513 was strictly dependent on prior growth on these substrates. A spontaneous regulatory variant of strain NA513 remained unable to utilize most of the mannityl opine analogs. Glutamine, mannose, and several analogs had no inhibitory effect on [14C]mannopine utilization by strain NA513.