The cyanomorpholino analog of doxorubicin (MRA-CN) is a potent cytotoxic agent which is known to cross-link DNA. A human ovarian carcinoma cell line, ES-2, was grown in increasing concentrations of MRA-CN from 0.1 to 0.5 nM. The resultant resistant subline, ES-2R, was 4-fold resistant to MRA-CN. DNA damage and repair in response to MRA-CN were compared in the parental and resistant cell lines using alkaline elution. DNA cross-links were detectable after 3-h incubation of the cells at 37 degrees C in MRA-CN at concentrations greater than or equal to 1.0 nM. Paradoxically, 2-fold more cross-links were detected in the ES-2R cells as compared with the ES-2 cells. This paradoxical difference in cross-links between the 2 cell lines was observed to increase with time of exposure to 2.5 nM of MRA-CN. Non-protein-associated DNA strand breaks were also detected in the 2 cell lines after exposure to 2.5 nM of the drug. The ES-2 cells consistently showed twice as many breaks as the ES-2R cells, which could explain the paradoxical higher apparent DNA cross-linking observed with the ES-2R cells after exposure to MRA-CN. Studies of the time course of cross-link repair after exposure to MRA-CN revealed that 75% of the DNA cross-links disappeared in the ES-2R cells by the end of 8 h in drug-free medium. In contrast, cross-links in the ES-2 cells were undetectable after 4 h, which coincided with a progressive increase in DNA strand breaks. The topoisomerase II level in the ES-2 cells was 2- to 4-fold higher than that in the ES-2R cells. However, proteinase K treatment of the lysed cells did not increase the number of apparent strand breaks produced by MRA-CN, suggesting that topoisomerase II may not be involved. These findings indicate that, in addition to DNA cross-linking, MRA-CN causes DNA strand breakage. Resistance to MRA-CN in the ES-2R cells is associated with more apparent DNA cross-linking and less DNA strand breakage, which may be a consequence of differences in DNA repair and/or nonspecific DNA degradation between the resistant and the sensitive cell lines.