Lentil (Lens culinaris Medik.) cultivars (Jordan 1 and Jordan 2) were sensitized to chlorsulfuron herbicide. Both cultivars were subjected to three doses of gamma-irradiation (90, 100 and 110 Gray) to develop plants tolerant/resistant to this herbicide. The herbicide-tolerant plants as well as the sensitized plants were subjected to acetolactate synthase (ALS) enzyme assay to reveal the biochemical basis of plant tolerance. The results indicated that as the radiation dose increased, the tolerance to the herbicide application decreased in both lentil cultivars. Cultivar Jordan 1 showed sensitivity to chlorsulfuron herbicide at all treatments compared with that of Jordan 2. ALS enzyme activity in the two lentil cultivars was inhibited by chlorsulfuron. The irradiated plants (90 Gray) in both treated seeds plants (M1) and seeds of tolerant plants (M2) showed a lower level of inhibition to high concentration (250 μg/l) of chlorsulfuron herbicide. The results suggested an alteration of the expression system of ALS gene(s) leading to overproduction of altered ALS enzyme at the herbicide binding site of the enzyme in lentil plant.