Gene therapy using the prodrug-activating enzyme P450 2B6 has shown substantial promise in preclinical and initial clinical studies with the P450 prodrugs cyclophosphamide and ifosfamide. We sought to optimize this therapy using the canine P450 enzyme 2B11, which activates cyclophosphamide and ifosfamide with Km of 80 to 160 micromol/L, approximately 10- to 20-fold lower than the Km of P450 2B6. Retrovirus encoding a P450 2B11-internal ribosome entry signal-P450 reductase expression cassette induced marked cyclophosphamide and ifosfamide cytotoxicity toward 9L gliosarcoma cells and exhibited an impressive bystander killing effect at micromolar prodrug concentrations, where P450 2B6 displayed low activity. Adeno-2B11, a replication-defective, E1/E3 region-deleted adenovirus engineered to coexpress P450 2B11 and P450 reductase, dramatically increased tumor cell-catalyzed cyclophosphamide 4-hydroxylation and cytotoxicity compared with Adeno-2B6 and effected strong bystander killing at low (20 micromol/L) cyclophosphamide concentrations. Further increases in cyclophosphamide cytotoxicity were obtained in several human cancer cell lines, including a 4-hydroperoxycyclophosphamide-resistant MCF-7 breast cancer cell line, when Adeno-2B11 was combined with Onyx-017, an E1b-55-kDa gene-deleted, tumor cell-replicating adenovirus that coamplifies and facilitates tumor cell spread of Adeno-2B11. To evaluate the therapeutic effect of P450 2B11 expression in vivo, 9L gliosarcoma cells transduced with P450-expressing retrovirus were grown as solid s.c. tumors in immunodeficient mice. Cyclophosphamide treatment on a metronomic, 6-day repeating schedule led to full regression of 9L/2B11 tumors but not P450-deficient control tumors, resulting in a tumor-free period lasting up to approximately 100 days. 9L/2B6 tumors regressed more slowly and exhibited a tumor-free period of only 21 to 39 days. Thus, P450 gene-directed enzyme prodrug therapy can be greatly improved by using the low Km P450 enzyme 2B11, which catalyzes intratumoral activation of cyclophosphamide and ifosfamide at pharmacologically relevant drug concentrations.