The synthesis, photophysics, and biochemical utility of a fluorescent NAD(+) analogue based on an isothiazolo[4,3-d]pyrimidine core (N(tz)AD(+)) are described. Enzymatic reactions, photophysically monitored in real time, show N(tz)AD(+) and N(tz)ADH to be substrates for yeast alcohol dehydrogenase and lactate dehydrogenase, respectively, with reaction rates comparable to that of the native cofactors. A drop in fluorescence is seen as N(tz)AD(+) is converted to N(tz)ADH, reflecting a complementary photophysical behavior to that of the native NAD(+)/NADH. N(tz)AD(+) and N(tz)ADH serve as substrates for NADase, which selectively cleaves the nicotinamides glycosidic bond yielding (tz)ADP-ribose. N(tz)AD(+) also serves as a substrate for ribosyl transferases, including human adenosine ribosyl transferase 5 (ART5) and Cholera toxin subunit A (CTA), which hydrolyze the nicotinamide and transfer (tz)ADP-ribose to an arginine analogue, respectively. These reactions can be monitored by fluorescence spectroscopy, in stark contrast to the corresponding processes with the nonemissive NAD(+).