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Ryegrass(Lolium multiflorum)is one of the most worrisome species with regard to herbicide resistance. This study aimed at characterizing the acetolactate synthase (ALS) enzyme activity of resistant biotypes (NC and AR) and susceptible biotypes to iodosulfuron-methyl-sodium (iodosulfuron). Different concentrations of pyruvate (1, 10, 20, 40, 60, 80 and 100 mM), of iodosulfuron (0 to 100 µM for the resistant biotypes and 0 to 0.5 µM for the susceptible biotype) and concentrations of valine, leucine and isoleucine (0, 0.001, 0.01, 0.1, 1, 10 and 100 mM) were used for the in vitro characterization. According to the results, the Michaelis constant (Km) values (pyruvate) for the assessed biotypes were similar. With iodosulfuron, resistant biotypes needed 395 to 779 times more herbicide in order to inhibit 50% of the enzyme activity, compared to the susceptible biotype. This confirmed a sensitivity modification of the ALS enzyme in the resistant biotypes. The resistant biotypes were less sensitive to the inhibition of the enzyme activity in the presence of the three amino acids. It was possible to conclude that biotype resistance was due to the enzyme insensitivity to iodosulfuron.