Human arterial smooth muscle grows in primary, as well as passaged, cultures with a heterogeneous pattern of monolayered regions alternating with multilayered with formation of tissue-like mounds or nodules. Cells in the monolayered region are large and well spread, whereas the multilayers are composed of smaller cells with poor spreading on tissue culture plastic. The two cell types were separated from passaged cultures of human arterial media. They were investigated with regard to capacity for attachment to tissue culture plastic, native collagen, and substrata coated with plasma fibronectin, the presence and distribution of cellular fibronectin and actin, as evaluated with specific staining, and the presence of cell-substratum contacts with interference reflection microscopy. The cells from monolayered regions were high adhesive and had abundant filamentous actin, often organized like stress fibers, numerous punctate and streak-like focal contacts with the substratum, and abundant fibronectin of which some was organized as streaks. In contrast, cells from multilayered regions were low adhesive, had very little filamentous actin, and had few or no stress fibers, fibronectin content was low and variable, and focal contacts with the substratum were poorly developed. The results demonstrate phenotypic heterogeneity in arterial smooth muscle cultures with expression toward a cell type with monolayered growth pattern or toward cells with a tendency for multilayered growth.