Interleukin 1β (IL-1β) and nitric oxide (NO) have potent growth-regulatory effects on different cell types. We found that epidermal growth factor–induced DNA synthesis in primary cultures of adult rat hepatocytes was inhibited by NO when it was provided by addition to the cultures of S-nitroso-N-acetyl-penicillamine (SNAP), an NO donor, as well as by addition of IL-1β in a dose-dependent manner. IL-1β also induced NO production and inducible NO synthase (iNOS) gene expression. The inhibition of DNA synthesis by IL-1β was completely abrogated when NO production was inhibited by N-monomethyl-l -arginine (NMA), a competitive inhibitor of iNOS. IL-1β–receptor antagonist (IL-1ra), which interferes with the interaction of IL-1β with target cells, also abolished the inhibitory effects of IL-1β on hepatocyte DNA synthesis as well as IL-1β–induced iNOS gene expression. We also found that hepatocyte DNA synthesis inhibition by IL-1β was completely antagonized by providing deoxynucleosides to bypass the block in ribonucleotide reductase, a rate-limiting step in DNA synthesis, thus implicating this enzyme in the mechanism of growth inhibition by IL-1β. These experiments extended prior observations on the growth-inhibitory actions of IL-1β on hepatocyte DNA synthesis, involving the IL-1β receptor, NO production, and ribonucleotide reductase.