Addition of protein kinase inhibitor H-7 leads to major changes in cell structure and dynamics. In previous studies [Citi, 1992: J. Cell Biol. 117:169-178] it was demonstrated that intercellular junctions in H-7-treated epithelial cells become calcium independent. To elucidate the mechanism responsible for this effect we have examined the morphology, dynamics, and cytoskeletal organization of various cultured cells following H-7-treatment. We show here that drug treated cells display an enhanced protrusive activity. Focal contact-attached stress fibers and the associated myosin, vinculin, and talin deteriorated in such cells while actin, vinculin, and N-cadherin associated with cell-cell junctions were retained. Furthermore, we demonstrate that even before these cytoskeletal changes become apparent, H-7 suppresses cellular contractility. Thus, short pretreatment with H-7 leads to strong inhibition of the ATP-induced contraction of saponin permeabilized cells. Comparison of H-7 effects with those of other kinase inhibitors revealed that H-7-induced changes in cell shape, protrusional activity, and actin cytoskeleton structure are very similar to those induced by selective inhibitor of myosin light chain kinase, KT5926. Specific inhibitors of protein kinase C (Ro31-8220 and GF109203X), on the other hand, did not induce similar alterations. These results suggest that the primary effect of H-7 on cell morphology, motility, and junctional interactions may be attributed to the inhibition of actomyosin contraction. This effect may have multiple effects on cell behavior, including general reduction in cellular contractility, destruction of stress fibers, and an increase in lamellipodial activity. It is proposed that this reduction in tension also leads to the apparent stability of cell-cell junctions in low-calcium medium.