A method combining electrophoresis and freeze-fracture electron microscopy is described; the method was used to determine the lateral diffusion coefficient of intramembrane particles (integral proteins) in the mitochondrial inner membrane. An electric current was passed through microsuspensions of purified, spherical inner membranes at pH 7.4, which caused an electrophoretic migration of intramembrane particles in the membrane plane into a single, crowded patch facing the positive electrode. The membrane microsuspensions were quick-frozen at specified times after the packed particles were released from the electrophoretic force and while the particles were diffusing back to a random distribution. Observed concentration gradients of intramembrane particles during this time were quantitatively compared with and found to follow a mathematical model for Fickian diffusion of particles on a spherical membrane. The results determine the kinetics of free diffusion of integral proteins at the resolution of individual proteins. The diffusion coefficient of the integral proteins in the mitochondrial inner membrane was determined to be 8.3 X 10(-10) cm2/sec at 20 degrees C, from which a root-mean-square displacement of 57 nm in 10 msec is predicted.