A variety of coenzymes that mediate various intermediary reactions have been shown to affect the self-splicing activity of primary transcripts of the T4 phage thymidylate synthase gene (td). Of all coenzymes examined, FMN was the most potent inhibitor and the order of inhibitory efficiency for coenzymes tested was as follows: FMN > thiamine pyrophosphate > NADP⁺ > NAD⁺ > coenzyme A > FAD > pyridoxal phosphate. The kinetic analysis showed that FMN and thiamine pyrophosphate act as competitive inhibitors whereas NADP⁺, NAD⁺>, coenzyme A, FAD, and pyridoxal phosphate act as noncompetitive inhibitors for the td intron RNA. The specificity of the splicing inhibition by competitive inhibitors was predominantly due to changes in Km so that increasing concentrations of guanosine overcame the suppression of the self-splicing activity. However, the splicing inhibition by noncompetitive inhibitors was not only due to changes in Km and kcat values but also Mg²⁺ concentration. The results suggest that coenzymes provide quite versatile picture of molecular actions in the self-splicing of the the td intron RNA.