Interleukin 1beta (IL-1beta) 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-1beta in a dose-dependent manner. IL-1beta also induced NO production and inducible NO synthase (iNOS) gene expression. The inhibition of DNA synthesis by IL-1beta was completely abrogated when NO production was inhibited by N-monomethyl-L-arginine (NMA), a competitive inhibitor of iNOS. IL-1beta-receptor antagonist (IL-1ra), which interferes with the interaction of IL-1beta with target cells, also abolished the inhibitory effects of IL-1beta on hepatocyte DNA synthesis as well as IL-1beta-induced iNOS gene expression. We also found that hepatocyte DNA synthesis inhibition by IL-1beta 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-1beta. These experiments extended prior observations on the growth-inhibitory actions of IL-1beta on hepatocyte DNA synthesis, involving the IL-1beta receptor, NO production, and ribonucleotide reductase.