Various hairy root clones were derived by transforming two kinds of Catharanthus roseus plants (Catharanthus roseus cvs. Little Linda and Little Delicata) with Agrobacterium rhizogenes ATCC 15834. Hairy root growth and indole alkaloid production were then investigated to select a high-yielding hairy root clone. Among three Amberlite resins (XAD-2, XAD-4, and XAD-7), XAD-7 greatly enhanced the release of catharanthine and ajmalicine from hairy root cultures, with an increase in total production. To enhance alkaloid production and secretion, a permeabilizing agent (dimethyl sulfoxide) and a fungal elicitor to provide physical and biochemical stress, respectively, together with in situ adsorption. Dimethyl sulfoxide (0.5% v/v) treatment with in situ adsorption using XAD-7 was found appropriate for releasing indole alkaloids from hairy roots without affecting cell viability. In addition, fungal elicitation by Penicillium sp. enhanced both alkaloid production and secretion. By combining in situ adsorption sequentially with these techniques, the release ratio of catharanthine and ajmalicine was enhanced up to 20 and 70%, respectively, which was 3.4 and 2 times higher than that obtained with in situ adsorption by XAD-7 alone. Over a 27-d culture period, the total amounts of catharanthine and ajmalicine produced were 67 and 30.15 mg/l, respectively. These results indicated that in situ adsorption sequentially applied with permeabilization and fungal elicitation have a synergistic effect on the production and secretion of indole alkaloids.