Protoplasts were isolated from Agrobucterium rhizogenes A-transformed cell line of Medicago sativa L. The highest yield of protoplasts (4.2×106per g fresh weight) was obtained from 12-d-old calluses after being subcultured on fresh medium. The viability of protoplasts reached over 80%. Protoplasts were induced to undergo sustained divisions when cultured in Durand et al. (DPD) medium supplemented with 2mgl-1 (9.05μM)2, 4-dichlorophenoxyacetic acid. 0.2mgl-1(0.93μM)kinetin, 0.3M mannitol, 2%(w/v)sucrose, and 500mgl-1 casein hydrolyzate at a plating density of 1.0×106perml. An agarose-beads culture method was appropriate for protoplast division of transformed alfalfa. The division frequency was ablut 30%. Numerous hairy roots were induced from protocalluses on Murashige and Skoog medium without grouwth regulators. Paper electrophoresis revealed that all of the regenerated hairy roots tested synthesized the corresponding opines. This protoplast culture system would be valuable for further somatic hybridization in forage legumes.