The use of precision-cut liver slices for toxicity and drug metabolism studies becomes more and more popular. So far, most of these studies are conducted using the dynamic organ culture system as incubation system. However, this system has some disadvantages, especially for the study of drug metabolism. Therefore, the aim of this study was to develop a simple incubation system for precision-cut liver slices. Rat liver slices were incubated individually in 12-well culture plates filled with 1.5 ml Krebs-Henseleit buffer, pH 7.4. Instead of glucose, fructose was used as carbohydrate source. The plates were put on a gyratory shaker (90 rpm) in a temperature controlled incubator (37 degrees C) under an atmosphere of 95% air/5% CO2. Under these conditions slices could be kept viable for at least 11 hr, which seems to be long enough for metabolism studies. Slice thickness was a critical factor in both studies on optimal incubation conditions and metabolism studies. A correlation was found between slice thickness (i.e. slice weight) and metabolite production (amount formed/mg slice) as demonstrated with tolbutamide and diazepam as test substances. It is demonstrated that a variation in slice thickness does not alter the number of cells involved in drug metabolism (i.e. the absolute amount of metabolite formed per slice does not alter). In conclusion, the way liver slices are incubated as well as the thickness of slices highly determines the results of studies on incubation conditions and metabolism studies with precision-cut liver slices.