Chemotaxis (CT) of cells is an important factor in the defense of organisms. For the study of this process several assays are available using the Boyden chamber. Its popularity, however, has led to a use of the system of which the basic qualities are not always known and so may lead to erroneous interpretation of results. Experiments were performed with the purpose of elucidating the significance of parameters regularly used in CT: 1. the cell count at the bottom of the permeable filter, 2. a chemotactic index, and 3. the leading front method. Neutrophils and eosinophils were stimulated with FMLP (10(-13)-10(-3) M). It appeared that: 1. the parameters are indicative only for (small) parts of the cell population, 2. for the comparison of effects of compounds complete concentration-effect relationships are needed, 3. to avoid cell to cell interactions a maximal cell purity is desired, and 4. if possible, no combinations of chemotactic agents should be tested.