Electron microscopy of thick (0.2-1.0 micron) sections of cytochalasin D-treated cells fixed in the presence of Ruthenium red revealed an extensive, surface-connected tubular compartment in HEp-2 cells. The tubules measured 120-220 nm in diameter and at least up to 6 microns in length. Morphometric analysis showed that in control cells about 0.2% of the total plasma membrane area (defined as all Ruthenium red-labeled membrane) appeared as vesicular or tubular profiles beneath the cell surface. However, after 15-30 minutes of cytochalasin D incubation about 4% of the total plasma membrane area is tubulated, and after 60-105 minutes as much as about 15% of the total plasma membrane appears as tubules. Clathrin-coated pits and caveolae-like structures were occasionally associated with the tubular membrane. Moreover, immunogold labeling showed that the tubular membrane contained transferrin receptors at about the same density as the nontubulated plasma membrane. Examination of cells in which endosomes and lysosomes were labeled with horseradish peroxidase before or after exposure to cytochalasin D showed that these organelles remained spherical, and that no horseradish peroxidase was present in the tubules. Moreover, the surface to volume ratio remained constant with increasing time of cytochalasin D incubation. Accordingly, the surface-connected tubules were not derived from endocytic structures but were formed by invagination of the plasma membrane. The tubule formation is reversible. When microtubules are depolymerized by nocodazole or colchicine treatment before the cells are exposed to cytochalasine D, tubule formation is strongly inhibited. Hence, the cytochalasin D-induced plasma membrane tubulation depends on intact microtubules.