We studied the effect of topical application of papaverine, a smooth-muscle relaxant, on the contraction of open, dorsal skin wounds in rats. Wound contraction was inhibited significantly in papaverine-applied wounds compared with saline-dressed control wounds. The in vitro effect of papaverine on wound contraction was studied by using human oral fibroblasts cultured three-dimensionally in the hydrated collagen gels containing papaverine. Papaverine inhibited collagen gel contraction in a dose-dependent manner. Any change in actin filament organization in fibroblasts cultured three-dimensionally in the collagen gels was observed by staining the filaments with fluorescent dye-conjugated phalloidin. In the control cultures, well-organized stress fibers were formed in the cell projections; in papaverine-treated fibroblasts, during the early stages of the treatment, the stress fibers were disrupted and actin filament aggregation was observed. In addition, papaverine induced a delay in the formation of the processes in fibroblasts cultured in collagen gels. These results indicate that wound contraction inhibition by papaverine is mediated by its effects on the organization of actin filaments of fibroblasts.