The normal contraction of open wounds and many forms of pathologic contracture are related by the presence of a contractile fibroblast known as a myofibroblast. The function of this cell has been postulated as a result of previous pharmacological, immunological, and biochemical testing on strips of contracted connective tissue. The purpose of this study was to develop a specific assay that could measure the concentration of one contractile element (actin) within cultured myofibroblasts isolated from a contracting wound and in normal fibroblasts from uninjured dermis. Rates of growth and actin concentration through 15 days of culture were compared among populations of paired control fibroblasts from normal dermis and granulating wound myofibroblasts from three patients. Growth curves showed that myofibroblasts always grew slower than fibroblasts. An enzyme-linked immunosorbent assay showed that actin concentration was generally greater in mass cultures of granulating wound myofibroblasts than in fibroblasts from uninjured dermis. During exponential growth (1-6 days) the average actin concentration among myofibroblast lines ranged from 24 to 62 pg/cell. Average actin levels among control fibroblasts ranged from 3 to 47 pg/cell during the same interval. After 15 days of culture, actin concentration peaked twice. The first actin peak occurred within the period of exponential growth. At confluency, cellular actin levels dropped. Superconfluent cultures exhibited a second actin peak that displayed an irregular pattern of actin concentration. The latter observation suggested an artifact that might be the result of three-dimensional matrix of cells that altered points of cell adhesion and produced an irregular pattern of actin concentration. These data show that the phenotype of increased actin in cultured myofibroblasts was carried over by myofibroblasts from contracted skin wounds to culture. Because of a higher concentration of actin in myofibroblasts than in undifferentiated fibroblasts, these data suggest that the differentiation process of myofibroblasts may be associated with an increased availability of monomeric actin for filament synthesis. This study demonstrates that the use of tissue culture and our enzyme-linked immunosorbent assay will be a useful method to study factors affecting myofibroblast phenotypic modulation. Future studies should be directed toward developing procedures for isolation of pure populations of myofibroblasts as well as extracellular matrices that would maintain the morphology of both differentiated myofibroblasts and normal undifferentiated fibroblasts.