A new and general electrophoresis method is described for the determination of dissociation constants of weak macromolecular complexes in the range of 10(-6) to 10(-4) M. The method is based on the measurement of the migration distance of a macromolecular complex in rapid dynamic equilibrium as a function of the interacting ligand concentration in a surrounding zone. Special advantages of the method are: its high sensitivity (dependent on the autoradiography, immunoblotting or staining technique used), its speed (electrophoresis time 20 min), and the independence of the Kd determination on the sample concentration of macromolecules. The latter is of great value for labile macromolecules: unknown partial inactivation does not influence the measurement. Studying the interactions between elongation factor EF-Tu and tRNA from E. coli we found for EF-Tu.GTP.aurodox.aminocyl-tRNA a Kd of 3 microM and for EF-Tu.GDP.aurodox.aminoacyl-tRNA a Kd of 11 microM at 9 degrees C.