In preliminary experiments, we found that 1H1, a monoclonal antibody directed against the v-src substrate cortactin, reacts with smooth muscle, myoepithelium, myofibroblasts, and manophages in fornaldehyde-fixed human tissues. To evaluate the use of this antibody as a diagnostic reagent, we tested the immunohistochemical distribution of cortactin in 61 mesenchymal neoplasms, II neuroectodermal neoplasms, and eight embryonal epithelial neoplasms. The results were compared with those obtained using antibodies against cu-smooth muscle actin and muscle-specific actin on a similar set of tissues. With the exception of positive staining in rhabdomyosarcoma, in this series only tumors with smooth muscle differentiation appeared to contain cortactin (16 of 19 leiomyosarcomas, one infantile fibrosarcoma, one malignant fibrous histiocytoma). Immunoelectron mircoscopy localized cortactin to the actin-associated dense bodies of the microfilament network. We conclude that cortactin may be a useful adjunct to alpha-smooth muscle actin and muscle-specific actin as a marker for the study and diagnosis of smooth muscle neoplasms and related lesions. Copyright (C) 1995 by W.B. Saunders Company