In HeLa and HEp2, cell lines derived from human carcinoma, application of cytochalasin D (CD) is followed in minutes by generalized cell contraction and zeiosis. Simultaneously, actin, myosin and tropomyosin, mostly from cables, become relocated in condensed masses. Most of these occupy the bases of the zeiotic knobs protruding at the cell surface. In contrast to most nontransformed cell types, in these cells both the protrusions and the contractile proteins are concomitantly translocated centripetally to the cell apex to form an aggregate of zeiotic knobs and a subjacent, membrane-associated, actin-based microfilamentous cap containing tropomyosin and myosin. The redistribution of these contractile proteins is antagonized by pretreatment with inhibitors of energy metabolism, or with cyclic adenine nucleotide. The centripetal movement appears to be guided by microtubules, which tend a radiate toward apical aggregates. Under the influence of CD 10 nm filaments associate in bundles. These processes are rapidly reversed after withdrawal of CD. The changes in distribution of actin, myosin and tropomyosin in relation to cell surface structures are considered in terms of the hypothesis that CD induces contraction of the microfilament apparatus of the cortex which, at least in part, remains tethered to plasma membrane.