Primary cultures of adult rat hepatocytes in a serum-free medium were observed by time lapse cinematography to proceed through mitotis and cytokinesis. An ultrastructural study of these cultures is presented with electron micrographs of each stage of mitosis and cytokinesis. The cultured hepatocytes begin to enter prophase about 48 hr after plating and proceed through mitosis in approximately 70 min not including cytokinesis. During this time, they remain somewhat flattened and joined to neighboring cells rather than rounding up. Both mononucleate and binucleate hepatocytes proceed through mitosis. Some mononucleate cells do not undergo cytokinesis, resulting in the formation of binucleate cells. In binucleate hepatocytes, both nuclei proceed through prophase simultaneously. Usually a single mitotic spindle with a large metaphase plate containing chromosomes from both nuclei is observed. Cytokinesis frequently occurs in binucleate hepatocytes which have a single mitotic spindle. Some binucleate cells form tripolar or 4-polar metaphase plates. In tripolar metaphases, some cells do not divide, resulting in multinucleate cells, whereas others undergo cytokinesis yielding three mononucleate cells or one binucleate and one mononucleate cell. Two mitotic spindles located perpendicularly to each other with microtubules intertwining have been observed in 4-polar metaphases. In this latter case, no cytokinesis has been observed. This study shows that both mononucleate and binucleate adult rat hepatocytes cultured in a serum-free medium in the presence of epidermal growth factor not only synthesize DNA, but progress through mitosis and often cytokinesis.