PURPOSE: Bendamustine has shown clinical activity in patients with disease refractory to conventional alkylator chemotherapy. The purpose of this study was to characterize the mechanisms of action of bendamustine and to compare it with structurally related compounds. EXPERIMENTAL DESIGN: Bendamustine was profiled in the National Cancer Institute in vitro antitumor screen. Microarray-based gene expression profiling, real-time PCR, immunoblot, cell cycle, and functional DNA damage repair analyses were used to characterize response to bendamustine and compare it with chlorambucil and phosphoramide mustard. RESULTS: Bendamustine displays a distinct pattern of activity unrelated to other DNA-alkylating agents. Its mechanisms of action include activation of DNA-damage stress response and apoptosis, inhibition of mitotic checkpoints, and induction of mitotic catastrophe. In addition, unlike other alkylators, bendamustine activates a base excision DNA repair pathway rather than an alkyltransferase DNA repair mechanism. CONCLUSION: These results suggest that bendamustine possesses mechanistic features that differentiate it from other alkylating agents and may contribute to its distinct clinical efficacy profile.