Expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene is induced by 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase. Synthesis of PEPCK mRNA is repressed by insulin, but remains detectable in H4IIE hepatoma cells exposed simultaneously to both 3-aminobenzamide and insulin. This capability of 3-aminobenzamide to block the inhibitory actions of insulin suggests that ADP-ribosylation is required for the regulation of PEPCK gene expression by insulin. Furthermore, neither changes in chromatin condensation nor cell growth status were linked to these events. The inability of 3,4-dihydro-5-methylisoquinolinone (PD128763), a selective inhibitor of poly(ADP-ribose) polymerase, to impede insulin-dependent repression of PEPCK gene expression, however, indicated that 3-aminobenzamide does not operate by inhibiting poly(ADP-ribosyl)ation. The potential involvement of mono(ADP-ribosyl)ation, a process that is also inhibited by 3-aminobenzamide, in the regulation of PEPCK gene activity was then evaluated. Analysis of poly(ADP-ribose) polymerase activity and poly(ADP-ribosyl)ation confirmed that there were no significant changes in response to insulin, while microsomal mono(ADP-ribosyl)transferase activity was elevated approximately fourfold. An increase in protein hydroxylamine-sensitive mono(ADP-ribosyl)ation was observed following insulin treatment. The sensitivity of the mono(ADP-ribosyl)transferase activity to 3-aminobenzamide but not PD128763 makes it plausible that mono(ADP-ribosyl)ation rather than poly(ADP-ribosyl)ation contributes to the regulation of PEPCK gene expression.