Cultured hepatocytes have been explored for use in a bioartificial liver. Spheroids formed by cultured hepatocytes exhibit enhanced liver‐specific functions. The kinetics of spheroid formation, using rat hepatocytes, was studied on positively charged surfaces that were either uncoated or coated with collagen or (hydroxyethyl)methacrylate (HEMA). Optimal spheroid formation was obtained on positively charged (Primaria) surfaces at inoculum densities in the range of (3–9) × 104 cells/cm2. Cells initially attached and spread out on the surface. Subsequent retraction led to the emergence of small clumps of cells attached to the surface, from which spheroids formed and shed off into suspension. The process of spheroid formation took more than 72 h and was accompanied by a decrease in the surface area occupied by attached cells. Optical sectioning of fluorescently stained spheroids using confocal microscopy indicated that most of the cells in the spheroid were viable. Spheroids also maintained a constant albumin synthesis rate for over 7 days in culture. Spheroid formation was evaluated in terms of the changes in spheroid diameter, the surface area covered by attached cells, and the total protein content of the fraction of cells that formed spheroids. The quantitative methodologies developed were used to assess the effect of inoculum cell concentration on spheroid formation and to evaluate the kinetics of spheroid formation on different surfaces both favorable and nonfavorable to spheroid formation.