We examined 4-hydroperoxycyclophosphamide (4HC) and phosphorodiamidic mustard (PM)-mediated cytotoxicity and DNA interstrand cross-link (ISC) induction in a 4HC-resistant subline, B5-180(3), that was derived from the subcloned human myeloid leukemia cell line KBM-7/B5. Based on the ratio of the 50% inhibitory concentration values, B5-180(3) cells were approximately 35-fold resistant to 4HC compared with the parental cells. 4HC-induced ISC levels (as measured by alkaline elution) were approximately 9-fold lower in the 4HC-resistant line than the parent line. To determine whether the approximately 9-fold reduction of initial 4HC-induced ISCs was related to elevated aldehyde dehydrogenase (ADH), we examined PM-mediated cytotoxicity and ISC induction in the two cell lines. Unlike 4HC, the cytotoxicity of PM is independent of ADH activity. B5-180(3) cells were between 5- and 7-fold resistant to the cytotoxic effects of PM and were only slightly (approximately 1.3-fold) resistant to PM-mediated ISC induction. Furthermore, when cells were exposed to 4HC in the presence of 40 micrograms/ml cyanamide, an efficient inhibitor of ADH, the resistance index based on the 50% inhibitory concentration values was decreased to approximately 8. Elevated ADH can therefore explain most of the decrease in 4HC-induced ISCs, with elevated GSH levels probably accounting for the remaining small increment of resistance. Thus, decreased levels of ISC induction can account for a factor of approximately 9 of the total approximately 35-fold resistance to 4HC. The remaining increment of resistance to 4HC, as well as the cross-resistance to PM, appears to relate to an enhanced ability of the resistant subline to tolerate drug-induced ISCs. Thus, multiple factors appear to be involved in the resistance of these cells to the cytotoxic and DNA-damaging effects of 4HC, with elevated ADH and temporal factors (related to the processing of DNA damage) being the most important of these.