We investigated the effects of the coenzyme NAD(+) (nicotinamide adenine dinucleotide) and its analogs on the self-splicing of primary transcripts of the phage T4 thymidylate synthase gene (td). Of all the nicotinamide coenzymes and analogs tested, NADP(+) was the strongest inhibitor, with at potency approximately threefold that of NAD(+). Kinetic analysis demonstrated that NAD(+) acts as a mixed type noncompetitive inhibitor for the td intron RNA with a K-i of 4.1 mM. The splicing specificity inhibition by NAD(+) is predominantly due to changes in K-m and k(cat), and was Mg2+ concentration dependent. The results suggest that both the ADP and nicotinamide moieties are the key structural features in NAD(+) responsible for the inhibition of splicing.