Effects of nicotinamide on expression of Cyp1a1 and Cyp1a2 genes were investigated in adult C57BL/6NCrj mouse hepatocytes in primary culture. The cells were cultivated for up to 8 days as monolayers or spheroids (multicellular aggregates), respectively, on collagen-coated or noncoated dishes. CYP1A1 mRNA was prominently induced after exposure to 3-methylcholanthrene (MCA) throughout the observation period with either type of culture. In monolayer-cultured cells, substantial induction of CYP1A2 mRNA by MCA was observed only at day 1 of cultivation, followed by a decrease to very low levels thereafter. In contrast, prominent elevation of both mRNA species in spheroid culture was observed throughout the 8-day experimental period. In the presence of nicotinamide, the decrease in expression of CYP1A2 mRNA in monolayer-cultured hepatocytes was restored concentration-dependently and at 10 mM nicotinamide the induced amounts were equivalent to those observed in spheroid cells. Nicotinamide also elevated CYP1A2 mRNA expression in spheroid culture, but in both cases induction of CYP1A1 mRNA decreased. Constitutive expression of CYP1A2 mRNA in monolayer culture rapidly decreased with increasing culture period, but addition of nicotinamide likewise inhibited the decrease. Nicotinamide also restored considerable expression of CYP1A2 mRNA when not present from the start of cultivation, but added 24 h before. Activities of aryl hydrocarbon hydroxylase (AHH) and acetanilide-4-hy-droxylase (AAH) were elevated with either type of culture after treatment with MCA and the presence of nicotinamide further increased the AAH, resulting in an increment in the activity ratio of AAH vs AHH. Intracellular contents of NAD were considerably elevated by addition of nicotinamide. However, isonicotinamide, which is not converted to NAD in the cell, also elevated CYP1A2 mRNA expression prominently. Furthermore, NAD contents in spheroid culture were not higher than those in monolayer culture. These observations indicate that the presence of nicotinamide in the medium restores constitutive and inducible expression of Cyp1a2 gene, independent of NAD, and thus the compound is useful for studying the underlying mechanism of regulation of this gene in adult mouse hepatocytes in monolayer culture.