Escherichia coli chorismate synthase (EC 4.6.1.4), purified aerobically, does not contain oxidised flavin mononucleotide (FMN) as judged by its uv/visible and fluorescence spectra. However, transient kinetic studies of functioning enzyme show that each enzyme tetramer binds four equivalents of FMNH₂, the reduced hydroquinone state. The higher affinity of chorismate synthase for FMNH₂ (Kₚ < 2 µM) relative to FMN (>20 µM) is similar to that of bacterial luciferase.