Cells of the H4-II-E line retain a number of the differentiated features of normal hepatocytes. Individual cultures contain both monolayer and stratified regions. Utilizing a variety of electron microscopic (EM) techniques, intercellular junctions and surface differentiations in both regions of the cultures were investigated. Gap junctions resembled those in normal liver, but were smaller and occurred infrequently. In situ fracturing methods revealed rectilinear arrays of particles in the apical (exposed) surfaces of the cultures. The tight junction forms varied in complexity ranging from linear fibrils to continuous, circular forms that appeared to occlude extracellular, spherical pockets between individual pairs of cells. The complex forms, particularly the continuous, circular forms, were more commonly found in stratified regions of the culture. Microvilli extended into the pockets, which probably correspond to structures seen in sections. The pockets excluded colloidal lanthanum and their membranes were strongly stained with the Wachstein-Meisel ATPase method.