Two clonal subpopulations of cells (termed A and D) obtained originally from a heterogeneous human colon adenocarcinoma were studied in vitro with regard to possible modification of responsivity to graded doses of doxorubicin (ADM) after long-term growth in medium containing the differentiating agent N-methylformamide (NMF). Non-NMF-adapted clone A and D cells exhibited biphasic response curves after exposure to graded doses of ADM (0-1.4 micrograms/ml, 1 hour, 37 degrees C). The inactivation slopes for the initial regions of the survival curves were 0.13 and 0.39 g/ml for clone A and D cells, respectively. NMF-adapted cells, however, exhibited decreased sensitivity to ADM killing as the inactivation slopes for the initial region of the survival curves increased to 0.26 and 0.59 g/ml for clone A and D cells, respectively. The final slopes of the biphasic response curves (doses above about 0.8 micrograms/ml) were not significantly different between clone A and clone D tumor cells in either the non-NMF- or NMF-treated conditions. These data on ADM responses after differentiation induction by NMF indicate that the combination of differentiation-inducing agents with certain chemotherapeutic agents may produce therapeutically undesirable effects and that preclinical data are necessary prior to implementation of possible combined-modality protocols.