We have used fluorescent analogue cytochemistry, image intensification, and digital image processing to examine the redistribution of alpha-actinin and vinculin in living cultured African green monkey kidney (BSC-1) cells treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Before treatment, microinjected alpha-actinin shows characteristic distribution along stress fibers and at adhesion plaques; vinculin is localized predominantly at adhesion plaques. Soon after the addition of TPA, highly dynamic membrane ruffles begin to form. These incorporate a large amount of alpha-actinin but little vinculin. Alpha-actinin is subsequently depleted, more or less uniformly, from stress fibers. Disrupted stress fibers often fragment into aggregates and move into the perinuclear region. Careful analyses of fluorescence intensity distribution indicate that alpha-actinin is depleted more rapidly from adhesion plaques than from stress fibers. Furthermore, the depletion of alpha-actinin from adhesion plaques is also faster than either the depletion of vinculin or the disappearance of focal contacts. These observations indicate that TPA may initiate disruption of stress fibers by interfering with a link between alpha-actinin and vinculin, causing alpha-actinin to be preferentially depleted from adhesion plaques.