Spreading of hepatocytes on different supports was examined using scanning electron microscopy. Positively charged Primaria plates gave a uniform morphology in 2 h. The spreading was rapid and the surface of the cells showed early prominent dips. The hepatocytes had one or two of these structures corresponding with nuclearity of the cells. The nuclear origin of the dips was confirmed after 6 h. The indentations contained solid structures the number, size, and shape of which were identical to the nucleoli seen by light microscopy. The spreading on the other supports was less uniform. Nuclear dips appeared more slowly and were less marked initially in their depths. The nuclear dipping was independent of cell density and took place under conditions under which the cells undergo phenotypic changes during culture. Individual phenotypic changes occur at different times and rates so that the initial signal for their onset cannot be determined with any certainty. However, the appearance of the dips was accompanied by DNA synthesis in the normally quiescent cells. The process stopped when the dipping was completed. The unavoidable change in nuclear morphology in spread cells may explain why maintenance of a spherical shape circumvents inappropriate DNA synthesis and maintains hepatocyte differentiation in vitro.