Although it is known that the depletion of cellular ATP induces a dramatic, reversible disruption of microfilament structures, the morphological pathway remains obscure. I have studied this process by following directly the dynamic redistribution of fluorescently labeled alpha-actinin and vinculin which had been microinjected into living mouse 3T3 fibroblasts. Before treatment, microinjected alpha-actinin displayed characteristic distribution along stress fibers, whereas vinculin was localized predominantly at adhesion plaques. The first response after adding NaN3 and 2-deoxyglucose was the retraction of lamellipodia, followed, over a period of 2 h, by a dramatic contraction of stress fibers and loosening of focal contacts. Vinculin plaques shrank from an elongated shape to small aggregates. During recovery, which was initiated by removing NaN3 and 2-deoxyglucose from the medium, lamellipodia appeared rapidly and alpha-actinin dispersed from contracted aggregates. Some partially dispersed aggregates later served as initiation sites for the formation of stress fibers. The recovery of vinculin plaques occurred predominantly through direct elongation, and focal contacts developed concomitantly. A small fraction of vinculin aggregates, however, moved into the perinuclear region without developing into adhesion plaques, and some new vinculin plaques formed de novo. Possible mechanisms involved and relationships to disruptions induced by other agents are discussed.