Expression of class 3 aldehyde dehydrogenase (ALDH-3) has been associated with acquired or inherent resistance to oxazaphosphorine (OAP) antineoplastic alkylating agents (eg. cyclophosphamide). We previously demonstrated that expression of transfected rat ALDH-3 can confer OAP-specific resistance in human MCF-7 cells (Bunting, K. D., Lindahl, R., and Townsend, A. J. (1994) J. Biol. Chem. 269, 23197-23203). However, the aldophosphamide intermediate inactivated by human class 1 ALDH (hALDH-1) has not proven to be a good substrate for the purified hALDH-3. We have examined the ability of transfected human or rat ALDH-3 to confer OAP resistance in V79/SDl cells. Clones expressing elevated human (386-5938 milliunits/mg) or rat (4-597 milliunits/mg, benzaldehyde/NADP+ substrate) ALDH-3 activity were 1.3- to 12-fold resistant to mafosfamide relative to control cells (<1 milliunit/mg). Resistance was correlated with hALDH-3 activity, and was reversed by pretreatment with the ALDH inhibitor diethylaminobenzaldehyde. Transfectants were cross-resistant to 4-hydroperoxycyclophosphamide and 4-hydroperoxyifosfamide but not to phosphoramide mustard, ifosfamide mustard, melphalan, or acrolein. DNA interstrand cross-links were reduced commensurately with the fold resistance to mafosfamide in the highest activity clone. A key finding was the detection of a metabolite, most likely carboxyphosphamide, that is formed only by cytosols from cells expressing either class 3 or class 1 ALDH.