Chorismate synthase, the seventh enzyme on the shikimate pathway, catalyzes the formation of chorismate from 5-enolpyruvylshikimate 3-phosphate (EPSP). This reaction involves the loss of phosphate from C(3) and hydrogen from the C(6) pro-R position of EPSP. In order to probe the mechanism of this reaction, [3-(3)H, 14C]EPSP has been synthesized and a secondary V/K tritium kinetic isotope measured for the reaction catalyzed by Neurospora crassa chorismate synthase. A small but significant value of kH/kT = 1.047 +/- 0.012 was observed. The reaction is also shown to be effectively irreversible. Previous experiments have measured a primary deuterium isotope effect on V/K at C(6) [Balasubramanian, S., Abell, C., & Coggins, J. R. (1990) J. Am. Chem. Soc. 112, 8581-8583], and there is additionally evidence in support of a flavin intermediate in the mechanism [Ramjee, M. N., Coggins, J. R., Hawkes, T. R., Lowe, D. J., & Thorneley, R. N. F. (1991) J. Am. Chem. Soc. 113, 8566-8567]. In the light of these observations the reaction mechanism probably involves cleavage of the C(6)-H and C(3)-O bonds in distinct but partially rate determining steps.