A cocktail of the following probe substrates for human drug-metabolizing enzymes was used to characterize hepatocyte preparations: phenacetin (for CYP1A2), diclofenac (CYP2C9), diazepam (CYP2C19), bufuralol (CYP2D6), midazolam (CYP3A4/5) and 7-hydroxycoumarin (for glucuronidation and sulphation). The cocktail was incubated with cryopreserved human, dog or minipig hepatocytes or with freshly prepared rat hepatocytes. Sample analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an Open Access environment that allowed less experienced MS operators to login, submit and analyse sample sets using predefined settings without the immediate attendance of an experienced analyst. Intrinsic clearances (CLint) were calculated from the disappearance of the compounds from the incubations. Initially, the cocktail used for human, rat and dog hepatocyte incubations contained 7-ethoxycoumarin instead of 7-hydroxycoumarin. However, 7-ethoxycoumarin had an inhibitory effect on the metabolism of phenacetin. The highest CLint estimated with human and dog hepatocytes was observed for 7-hydroxycoumarin. For rat and minipig hepatocytes, the highest CLint was observed for bufuralol. In incubations with dog and minipig hepatocytes, the lowest CLint was seen with diclofenac, whereas for human and rat hepatocytes, the lowest value was observed with diazepam and phenacetin, respectively. When the cocktail was incubated together with human hepatocytes and 1 microM ketoconazole, the CLint of midazolam was decreased to about 7.5% of the control value, whereas the metabolism of the other cocktail compounds was virtually unaffected by this CYP3A inhibitor. It is suggested that a cocktail of specific human probe substrates for drug-metabolizing enzymes can be used routinely for the determination of the metabolic capacity of hepatocyte preparations in order to ensure the quality and reproducibility of experiments. Moreover, a cocktail of specific probe substrates can also be a useful tool for studies on enzyme inhibition.