Abstract: Aims/Background: We report in this study that, 10 mM nicotinamide can stimulate the proliferation of primary rat hepatocytes in serum-free Dulbecco's modified Eagle's medium supplemented with 10 ng/ml epidermal growth factor and that small hepatocyte colonies appear from 4 to 5 days after plating. We examined the effects of nicotinamide-related agents on the growth and differentiation of primary rat hepatocytes and on the appearance of small hepatocyte colonies. Methods: As nicotinamide is an aqueous vitamin named niacin and known to act as an inhibitor of poly (ADP-ribose) polymerase (PARP), we therefore chose to examine the effects on hepatocytes of three nicotinamide-related agents, nicotinic acid (NA) which is also a niacin, 3-aminobenzamide (3-AB) which is a strong inhibitor of PARP but is not a niacin, and 3-acetylpyridine (3-AP) which is a weak inhibitor of PARP and also not a niacin. To examine their effects on the growth of the cells and on the formation of the colony, immunocytochemistry for BrdU was carried out. Expression of albumin, tryptophan 2,3-dioxygenase (TO), and connexin 32 (Cx32) mRNAs were used as marks of hepatic differentiation. Intracellular NAD+ content was also measured. Results: At concentration of 10 mM, NA could not enhance the proliferation of mature hepatocytes but induced the appearance of small hepatocyte colonies. At concentration of 5 mM, 3-AB enhanced the proliferation of the hepatocytes but did not induce small hepatocyte colonies. On the other hand, although 10 mM 3-AP remarkably inhibited the DNA synthesis of the cells, the expression not only of albumin but also of TO and Cx32 mRNAs in the cells was well maintained for more than one week. The intracellular NAD+ concentration was correlated with the proliferation of the hepatocytes. Conclusion: These results suggest that the intracellular NAD+ content may be correlated with the proliferation of primary hepatocytes and that the supplementation of niacin in the medium may be important for the appearance of small hepatocyte colonies.