Human mammary carcinoma cell line MCF-7 cells grown on type I collagen gels floating in a medium occasionally invaginated into the gels as a cell mass and formed cylindrical or domed structures within it. The 0.05% Triton-insoluble cytoskeleton of such cellular structures sedimented as a white flocculent layer at the boundary between 60 and 70% sucrose layers by ultracentrifugation, and consisted of 4 basal components: 54-kD (beta-tubulin), 45-kD, 42-kD (actin), and 39-kD polypeptides. By contrast, the isolated cytoskeleton of MCF-7 cells grown as monolayers on plastic substratum formed a finer cytoskeletal network with a smaller buoyant density and consisted of two distinct polypeptides with apparent molecular sizes of 80-kD and 65-kD in addition to the 4 basal components found in the morphologically developing cells. The present results indicate that the cytoskeleton of MCF-7 cells forming the three-dimensional cellular structures within collagen gels is lacking in these two polypeptides, and that it has a coarser cytoskeletal network with a greater buoyant density than that of the monolayered cells on plastic.