Primary cell cultures of human myofibroblasts can be obtained from desmoplastic breast carcinoma, a tissue rich in these cells. Explants of infiltrating breast carcinoma (scirrhous type) give rise to outgrowths of cells which are predominantly (greater than 90%) myofibroblasts. These cells retain their identifying morphologic characteristics in cell culture by both ultrastructural and immunocytochemical criteria: the presence of elongated cells with longitudinally oriented thin filaments peripherally condensed to form dense zones and abundant rough endoplasmic reticulum; cytoplasmic immunoreactivity for myosin and Type V collagen. These myofibroblasts continue to divide and retain their morphologic characteristics for at least 14 days. These findings allow for further in vitro studies concerning both the role and regulation of the myofibroblast in the desmoplastic response.