Primary rat hepatocytes can self-assemble to form multicellular spheroids when plated onto Primaria petri dishes or suspended in stirred vessels. These spheroids exhibit prolonged viability, enhanced liver-specific functions and differentiated ultrastructure compared to monolayer cultures. Upon transfer to collagen coated surface, or upon the addition of fetal bovine serum (FBS) to the culture, these spheroids began to disassemble and spread on the surface. The dynamics of cytochrome P450 CYP1A1/2 activity in the course of spheroid disassembly was examined in situ by detection of the fluorescent product, resorufin, of ethoxyresorufin O-dealkylation. Optical sectioning of the disassembling spheroids by confocal microscopy demonstrated that hepatocytes that reverted to monolayer exhibited markedly lower CYP1A1/2 activity than those that remained in a multilayered structure. This occurred whether the disassembly was caused by incubation with FBS-containing medium or by cultivation on a collagen-coated surface. When spheroids were cultured on the surface of agar, the disassembly process was retarded even in the presence of FBS. However, even in those intact spheroids, the exposure to FBS markedly decreased CYP1A1/2 activity. The decreased CYP1A1/2 activity was correlated to a diminished smooth endoplasmic reticulum as seen in the transmission electron micrograph. The results clearly demonstrate that the disassembly of hepatocyte spheroids led to decreased CYP1A1/2 activity. Furthermore, FBS contained a factor that caused CYP1A1/2 to decrease even in intact spheroids.