Electrophoretic light scattering (laser Doppler electrophoresis) has been employed to study the effects of concanavalin A (Con A) and succinyl-Con A on the electrophoretic mobility distribution of resident guinea pig peritoneal macrophages. Con A, a tetrameric lectin, decreases slightly the mean mobility and increases by approximately 3-fold the width of the electrophoretic mobility distribution of resident macrophages. This effect can be abolished by alpha-methyl-D-mannoside, a hapten sugar of Con A. These effects were present in both low (0.010 M) and high (physiological, 0.15 M) ionic-strength media. Since lower ionic strengths correspond to a larger Debye screening distance, these data suggest that the alterations in the electrophoretic mobility distribution are not restricted to the outer portion of the glycocalyx. Succinyl-Con A, a dimeric derivative, was found to have no effect on the mobility distribution. However, the mean mobility decreased and the width increased over 4-fold when succinyl-Con A-treated macrophages were exposed to anti-Con A. These observations indicate that cross-linking of Con A receptors is an important process in the electrokinetic alterations of the macrophage surface. These results may have important consequences for the elucidation of the details of the endocytic mechanism.