Antibodies to skeletal muscle actin were produced in rabbits and purified by affinity chromatography. Direct labeling of SDS-PAGE gels of whole cell homogenates from mouse fibroblast cells showed that actin was the only protein detected by these antibodies. Using this immunospecific reagent, we localized actin in cultured fibroblasts using the EGS fixation-permeabilization procedure with the ferritin bridge labeling technique. Swiss 3T3-4 mouse fibroblasts were chosen as an example of highly adherent untransformed cells with prominent microfilament bundles, and L929 mouse fibroblasts were chosen as an example of poorly adherent, rounded, transformed cells with prominent microvilli. Using these two cell types, we have characterized the intracellular distribution of action. Actin was only detected in locations in which morphologically recognizable 60 A microfilaments were found. By both fluorescence and electron microscopy, actin was found in surface ruffles, microvilli, microfilament bundles, the microfilament mat, and the leading lamellae of Swiss 3T3 and L929 cells. In addition, actin was found surrounding micropinosomes and macropinosomes. On the other hand, there was no actin detected around the base of coated pits. Morphometric quantitation showed that almost all the actin was localized in microfilamentous structures. Our results suggest that actin has an important role in cell motility and adhesion, and in the endocytosis of pinosomes, but that actin may not be involved in intracellular processes such as saltatory motion of intracellular organelles.