Abstract The mechanism by which phenylalanine ammonia-lyase (PAL, EC 184.108.40.206) catalyzes the reversible elimination of ammonia from phenylalanine yielding (E)-cinnamic acid has gained much attention in the recent years. Dehydroalanine is essential for the catalysis. It was assumed that this prostetic group acts as the electrophile, leading to a covalently bonded enzyme–intermediate complex with quarternary nitrogen of phenylalanine. Recently, an alternative mechanism has been suggested in which the enzyme–intermediate complex is formed in a Friedel–Crafts reaction between dehydroalanine and orthocarbon of the aromatic ring. Using semiempirical calculations we have shown that these two alternative mechanisms can be distinguished on the basis of the hydrogen secondary kinetic isotope effect when tritium label is placed in the orthopositions. Our calculations indicated also that the kinetic isotope effect measured using ring-labeled d 5-phenylalanine could not be used to differentiate these alternative mechanisms. Measured secondary tritium kinetic isotope effect shows strong dependence on the reaction progress, starting at the inverse value of k H/ k T = 0.85 for 5% conversion and reaching the normal value of about 1.15 as the conversion increases to 20%. This dependence has been interpreted in terms of a complex mechanism with initial formation of the Friedel–Crafts type intermediate.