The use of human hepatocytes in drug metabolism studies overcomes the difficulty in extrapolating from animal data. As drug metabolites are often involved in pharmacological activity and toxicity, it is essential to determine these compounds early in the development of a drug. We previously demonstrated that mitoxantrone metabolism exhibits a great interspecies variability: the two main metabolites in humans being dicarboxylic acid (the major metabolite) and monocarboxylic acid. In this paper we analyse their respective cytotoxicities with two endpoint tests: neutral red uptake (cell viability) and MTT reduction (cell metabolism) after a 24- or 48-hr exposure. Results demonstrated less or no toxicity of mono- and dicarboxylic acid derivatives compared with mitoxantrone. In vitro studies using human hepatocytes are scarce because of the limited availability of human livers. After developing a new method for cryopreservation of human hepatocytes, we evaluated them before and after cryopreservation using erythromycin as a test molecule. The cell viability test was more reproducible than the cell metabolism test after 38 hr of cell seeding.