The distribution of a major glycoprotein (fibronectin) of human fibroblast cultures was studied in immunoelectron microscopy with peroxidase- or ferritin-labeled antibodies. External fibronectin was visualized in pericellular structures, in some areas on the growth substratum, and to a lesser degree in close association with the upper and lower surface membranes of the cell. The pericellular fibronectin-containing structures consisted of amorphous or vaguely fibrillar material forming strands or patches, 50-500 nm in diameter; the structures appeared to mediate distant cell-to-cell and cell-to-substrate contacts. When in close association with the plasma membrane, fibronectin markers were seen as discrete patches. The exact relationship between this form of fibronectin and the plasma membrane, however, remained open. Filamentous material was commonly seen in the cortical cytoplasm under patches of membrane-associated fibronectin. The distribution that we observed is consistent with the proposed roles of fibronectin in cell interactions with neighboring structures and with its presence in vivo as an extracellular glycoprotein in connective tissue matrix and basal laminae.