Chemical studies of the Chinese herb Corydalis saxicola Bunting led to the isolation and identification of 14 alkaloids, 1-14. Seven of these compounds, 4-9 and 11, were obtained from this plant for the first time. Feruloylagmatine (7) is the first guanidine-type alkaloid to be identified in the family Papaveraceae and in dicotyledonous plants. All of the isolated compounds were assayed for inhibitory activity against human DNA topoisomerase I. A DNA cleavage assay demonstrated that these alkaloids specifically inhibit topoisomerase through stabilization of the enzyme-DNA complex. Among the isolated alkaloids, (-)-pallidine (8) and (-)-scoulerine (11) showed strong inhibitory activities toward topoisomerase I that were comparable to camptothecin, a typical topoisomerase I inhibitor. A preliminary structure-activity relationship study suggested that the quaternary ammonium ion might play an important role in topoisomerase I inhibition by the isoquinoline alkaloids. These data indicated that DNA topoisomerase I inhibition represents probably one of the anticarcinogenic mechanisms of C. saxicola.