Hepatocytes can self-assemble into spheroids, which express enhanced differentiated function and may provide an important tool in tissue engineering. This study examines cell junction components as hepatocytes form either monolayer or spheroid, and correlates morphological changes with cytochrome P-450 function. Rat hepatocytes were cultured on either high- density (1 μg/cm2) or low-density (1 ng/cm2) fibronectin, which promotes monolayers or spheroids, respectively. Immunofluorescence demonstrates that the cell-cell adhesion receptor cadherin localizes to cell borders in monolayers and aggregates. In contrast, actin cytoskeleton differs dramatically between the two morphologies. Actin and β-catenin, which links cadherins to actin, colocalize in aggregates to a greater extent than in monolayers. The activity of cytochrome P-450 was measured in situ by determining the level of the fluorescent compound, resorufin, a product of P- 450-mediated O-dealkylation of 7-ethoxyresorufin. As cells aggregate into spheroids, resorufin increases, with spheroids demonstrating at least fivefold higher fluorescence than monolayers. Spheroids placed on primaria plates in the presence of serum disassemble into monolayers, with simultaneous decrease in fluorescence as cells develop a flattened morphology. Thus, we report findings that demonstrate a correlation between in vivo-like morphology and specific cytochrome P-450 function.