The H-35 rat hepatoma is relatively insensitive to the anthracycline antibiotic, daunorubicin (DNR), requiring 0.25 microM daunorubicin for inhibition of cell proliferation by 50%. Studies were undertaken to investigate the basis for the apparent intrinsic resistance in this cell line. The relative insensitivity of the H-35 cells to DNR is not a function of metabolic inactivation of DNR to deoxyaglycone derivatives; after a 2-h incubation, less than 10% of drug is metabolized, exclusively by conversion to daunorubicinol. The limited toxicity of DNR to the rat hepatoma may be explained, in part, by the absence of DNA strand breaks at daunorubicin concentrations up to 1 microM while higher (supraclinical) DNR concentrations (5 and 10 microM) produce direct, "non-protein-associated" DNA strand breaks. Limited daunorubicin toxicity in this tumor cell line may also be related to the apparent absence of free radical-mediated cellular damage as the free radical scavengers dimethyl sulfoxide, catalase, methanol, and mannitol fail to reverse the inhibitory effect of 1 microM DNR on cell proliferation. Daunorubicin does not induce leakage of the cytoplasmic enzyme, glutamic oxaloacetate transaminase, or diminish mitochondrial enzyme function. Conversely, while drug effects on RNA synthesis are small, and protein synthesis is minimally diminished, inhibition of cell proliferation corresponds closely with inhibition of DNA synthesis.