We present a detailed protocol for the transient transfection of non-proliferating primary cultured hepatocytes that is easily reproducible. Using a modification of the classical CaPO4/DNA precipitation method, this protocol is an inexpensive alternative to other methods that are often cumbersome, expensive, difficult to reproduce or harmful to primary hepatocytes. Because only 0.5 x 10(6) cells are needed for a single transfection experiment, several reporter genes can be introduced into hepatocytes of a single liver preparation. With our protocol, different plasmids can be introduced into one cell. In this way, cis-trans interactions can be examined and reporter gene expression can be normalized for transfection efficiency. Furthermore, we describe details of a transfection experiment with two different reporter gene vectors using a luciferase gene and a lacZ gene. The results presented may be helpful to other groups concerned with improved timing of transfection experiments.