We have characterized the kinetics and substrate requirements of prenyl-flavin synthase from yeast. This enzyme catalyzes the addition of an isopentenyl unit to reduced flavin mononucleotide (FMN) to form an additional six-membered ring that bridges N5 and C6 of the flavin nucleus, thereby converting the flavin from a redox cofactor to one that supports the decarboxylation of aryl carboxylic acids. In contrast to bacterial enzymes, the yeast enzyme was found to use dimethylallyl pyrophosphate, rather than dimethylallyl phosphate, as the prenyl donor in the reaction. We developed a coupled assay for prenyl-flavin synthase activity in which turnover was linked to the activation of the prenyl-flavin-dependent enzyme, ferulic acid decarboxylase. The kinetics of the reaction are extremely slow: kcat = 12.2 ± 0.2 h-1, and KM for dimethylallyl pyrophosphate = 9.8 ± 0.7 μM. The KM for reduced FMN was too low to be accurately measured. The kinetics of reduced FMN consumption were studied under pre-steady state conditions. The reaction of FMN was described well by first-order kinetics with a kapp of 17.4 ± 1.1 h-1. These results indicate that a chemical step, most likely formation of the carbon-carbon bond between C6 of the flavin and the isopentenyl moiety, is substantially rate-determining in the reaction.