Adenylated dinuclectides (Ap(n)A) are regulatory molecules that control various cellular processes. A very likely intracellular target for Ap(4)A are enzymes that require ATP as either substrate or modulator. We report the results of new biochemical studies aimed at characterizing the Ap(4)A interaction with firefly luciferase, by using the luminometric and thin layer chromatography techniques. The data presented herein demonstrate that Ap(4)A is a noncompetitive inhibitor for the ATP-induced luminescence. These results together with our previous findings that Ap(4)A is a luciferase substrate [Nucleosides Nucleotides Nucleic Acids 23 (2004) in press.] support the notion that, similar to its interaction with P-2 receptors, Ap(4)A also has a dual interaction with luciferase. Other Ap(n)As (n = 2, 5, and 6) also inhibited the ATP-luciferase interaction. Since Ap(n)As may have similar interactions with other intracellular ATP-requiring enzymes, the study presented herein validates ulterior investigations of the Ap(a)A interaction with such enzymes, and opens the way to a better understanding of their intracellular roles. (C) 2004 Elsevier Inc. All rights reserved.