A procedure for the systematic regeneration from Ajuga hairy root was developed. Supplementation of an auxin, naphthaleneacetic acid (0.1 mg/l), in the hairy root growth culture enhanced the cell growth rate. Mechanical fragmentation for 10 s in a commercial blender enabled a large number of root fragments to be produced efficiently. Supplementation of a cytokinin, benzyladenine (10 mg/l), exhibited the highest plantlet formation frequency in the plantlet formation stage. The plantlets longer than 4 mm in length were able to grow into whole plants on agar containing modified MS medium. Ajuga hairy root in which the beta-glucuronidase (GUS) gene was introduced under control of the tomato ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (rbcS3B) promoter was constructed by the Agrobacterium rhizogenes-mediated co-transformation. The GUS-transformed hairy root could also be efficiently regenerated into plantlets through this procedure. GUS activity was detected in leaf tissue of the regenerated plants.