ADP-ribosyl cyclases catalyze the transformation of nicotinamide adenine dinucleotide (NAD+) into the calcium-mobilizing nucleotide second messenger cyclic adenosine diphosphoribose (cADP-ribose) by adenine N1-cyclization onto the C-1' ' position of NAD+. The invertebrate Aplysia californica ADP-ribosyl cyclase is unusual among this family of enzymes by acting exclusively as a cyclase, whereas the other members, such as CD38 and CD157, also act as NAD+ glycohydrolases, following a partitioning kinetic mechanism. To explore the intramolecular cyclization reaction, the novel nicotinamide 2-fluoroadenine dinucleotide (2-fluoro-NAD+) was designed as a sterically very close analogue to the natural substrate NAD+, with only an electronic perturbation at the critical N1 position of the adenine base designed to impede the cyclization reaction. 2-Fluoro-NAD+ was synthesized in high yield via Lewis acid catalyzed activation of the phosphoromorpholidate derivative of 2-fluoroadenosine 5'-monophosphate and coupling with nicotinamide 5'-monophosphate. With 2-fluoro-NAD+ as substrate, A. californica ADP-ribosyl cyclase exhibited exclusively a NAD+ glycohydrolase activity, catalyzing its hydrolytic transformation into 2-fluoro-ADP-ribose, albeit at a rate ca. 100-fold slower than for the cyclization of NAD+ and also, in the presence of methanol, into its methanolysis product beta-1' '-O-methyl 2-fluoro-ADP-ribose with a preference for methanolysis over hydrolysis of ca. 100:1. CD38 likely converted 2-fluoro-NAD+ exclusively into the same product. We conclude that A. californica ADP-ribosyl cyclase can indeed be classified as a multifunctional enzyme that also exhibits a classical NAD+ glycohydrolase function. This alternative pathway that remains, however, kinetically cryptic when using NAD+ as substrate can be unmasked with a dinucleotide analogue whose conversion into the cyclic derivative is blocked. 2-Fluoro-NAD+ is therefore a useful molecular tool allowing dissection of the kinetic scheme for this enzyme.