Monoclonal antibody (MAb) F7-26 generated against nitrogen mustard (HN2)-treated DNA (O.S. Frankfurt, Exp. Cell Res., 170: 369-380, 1987) reacted with regions of local DNA denaturation (distortion) induced by DNA alkylation. The relationship between immunoreactivity of cellular DNA with MAb F7-26 and cytotoxic effects of HN2, L-phenylalanine mustard (L-PAM), and 1,3-bis(2-chloroethyl)-1-nitrosourea was studied in HeLa S3 cultures. Cells were treated with drugs for 1 h and assessed for cell survival by colony formation assay and for DNA immunoreactivity by flow cytometry. Cells were fixed in ethanol, exposed to MAb, and stained with fluorescein-labeled anti-mouse immunoglobulin. Immunofluorescence (IF) intensity was measured on a flow cytometer. For each drug the cell killing and the binding of MAb to DNA appeared in the same dose ranges. A strong correlation (r = 0.96) between cell survival (log10 surviving fraction) and IF was observed when data for HN2, L-PAM, and 1,3-bis(2-chloroethyl)-1-nitrosourea were combined. This correlation was apparent in the range of 1-5 log10 cell killing. Enhancement of L-PAM cytotoxicity by buthionine sulfoximine (BSO) or hyperthermia was accompanied by a proportional increase of DNA immunoreactivity with MAb F7-26. The enhancement factors calculated from survival curves (a ratio of the dose decreasing cell survival by 1 log10 for L-PAM alone to that for L-PAM combined with modulating factor) were 1.67, 1.58, and 3.07 for BSO, hyperthermia, and BSO plus hyperthermia, respectively. For the same treatment regimens the enhancement factors calculated from drug dose-IF curves were 1.73, 1.34, and 3.79. A strong correlation between log10 surviving fraction and IF intensity (r = 0.93) was observed when data for L-PAM alone or L-PAM combined with BSO and/or hyperthermia were considered together in the range of 1-6 log10 cell killing. The cytotoxicity of alkylating agents and nitrosoureas and the effectiveness of factors modulating chemotherapeutic effects can be predicted by flow cytometry analysis of DNA immunoreactivity with MAb F7-26.