Previous studies have shown that Adriamycin can react with formaldehyde to yield an activated form of Adriamycin that can further react with DNA to yield Adriamycin-DNA adducts. Because hexamethylenetetramine (HMTA) is known to hydrolyze under cellular conditions and release six molecules of formaldehyde in a pH-dependent manner, we examined this clinical agent for its potential as a formaldehyde-releasing prodrug for the activation of Adriamycin. In IMR-32 neuroblastoma cells in culture, increasing levels of HMTA resulted in enhanced levels of Adriamycin-DNA adducts. These adducts were formed in a pH-dependent manner, with 4-fold more detected at pH 6.5 compared with pH 7.4, consistent with the known acid lability of HMTA. The resulting drug-DNA lesion was shown to be cytotoxic, with combined Adriamycin and prodrug treatment resulting in a 3-fold lower IC(50) value compared with that of Adriamycin alone. Given the acidic nature of solid tumors and the preferential release of formaldehyde from HMTA in acidic environments, HMTA therefore has some potential for localized activation of Adriamycin in solid tumors.