P140K-MGMT and G156A-MGMT genes encode two O(6)-benzylguanine-resistant O(6)-alkylguanine DNA alkyltransferase proteins that confer a high degree of O(6)-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or O(6)-benzylguanine and temozolomide resistance to primary hematopoietic cells. In this study, we directly compared these and three other O(6)-benzylguanine-resistant MGMT genes for their ability to protect the human erythroleukemia cell line, K562, using a direct competitive selection strategy to identify the mutation that conferred the greatest degree of protection from O(6)-benzylguanine and either BCNU or temozolomide. MFG retroviral vector plasmids for each of these mutants [G156A-MGMT (ED(50) for O(6)-benzylguanine, 60 micromol/L); and P140K-MGMT, MGMT-2 (S152H, A154G, Y158H, G160S, L162V), MGMT-3 (C150Y, A154G, Y158F, L162P, K165R), and MGMT-5 (N157T, Y158H, A170S; ED(50) for benzylguanine, >1,000 micromol/L)] were mixed, and the virus produced from Phoenix cells was transduced into K562 cells. Stringent selection used high doses of O(6)-benzylguanine (800 micromol/L) and temozolomide (1,000 micromol/L) or BCNU (20 micromol/L) administered twice, and following regrowth, surviving clones were isolated, and the MGMT transgene was sequenced. None of the mutants was lost during selection. Using temozolomide, the enrichment factor was greatest for P140K-MGMT (1.7-fold). Using BCNU selection, the greatest enrichment was observed with MGMT-2 (1.5-fold). G156A-MGMT, which is the least O(6)-benzylguanine-resistant MGMT gene of the mutants tested, was not lost during selection but was selected against. The optimal mutant MGMT useful as a drug resistance gene may depend on whether a methylating or chloroethylating agent is used for drug selection.