This investigation compared the effects of hydroxymethylacylfulvene (HMAF), a novel antitumor drug with alkylating properties, in eight human tumor (prostate, colon, and leukemia) cell lines, and five human normal (prostate and renal proximal tubule epithelial, colon mucosa, fibroblasts, and endothelial) cell lines. Drug-induced growth inhibition paralleled the uptake of HMAF into both tumor and normal cells, although normal cells were 3- to 4-fold more tolerant to the accumulated drug. In both tumor and normal cells, approximately two-thirds of internalized [(14)C]HMAF-derived radioactivity was bound covalently to macromolecules. Trypan blue exclusion and cell counts indicated that HMAF was cytotoxic in tumor but cytostatic in normal cells. Correspondingly, profound apoptosis was detected in all tumor cell lines examined. A 4-hr treatment with HMAF followed by 20-hr post-incubation induced a potent DNA fragmentation in nearly all tumor lines. Apoptosis-resistant PC-3 and HT-29 cells underwent significant DNA fragmentation after 24 hr of continuous treatment with HMAF. In contrast to tumor cell lines, marginal or very low levels of apoptosis were detected in the normal cells even after prolonged treatments with HMAF at concentrations that exceeded 15- to 800-fold the GI(50) values in tumor cells. This resistance of normal cells to apoptosis could not be accounted for by differences in drug accumulation or drug covalent binding to macromolecules. The qualitatively different responses of the tumor and normal cells studied suggest a greater tolerance of normal cells to HMAF-macromolecular adducts. The demonstrated differential cytotoxic/cytostatic and apoptotic effects of HMAF can be of significance for the clinical use of this promising new agent.