Cultures of Nicotiana tabacum cells homozgous for a mutation (S4) at the SuRB locus that confers resistance to the sulfonylurea herbicides chlorsulfuron and sulfometuron methyl (Chaleff and Ray 1984; Chaleff and Bascomb 1987) were used to isolate a doubly mutant cell line (S4 Hra/S4+) resistant to even higher herbicide concentrations. Growth of cells homozygous for both the S4 and Hra mutations (S4 Hra/S4 Hra) was uninhibited by a herbicide concentration 500-fold higher than a concentration by which growth of S4+/S4+ callus was inhibited by 75%. Plants homozygous for both mutations were at least five-fold more resistant to foliar applications of chlorsulfuron than were singly mutant S4+/S4+ plants. This enhanced resistance was inherited as a single, semidominant, nuclear trait that is genetically linked to the S4 mutation. Acetolactate synthase (ALS) activity in extracts of leaves of doubly mutant (S4 Hra/S4 Hra) plants was approximately 20-fold more resistant to inhibition by chlorsulfuron and sulfometuron methyl than was ALS activity in singly mutant (S4+/ S4+) leaf extracts, which was in turn more resistant to inhibition by these compounds than was the normal enzyme. Extracts prepared from plants of these three genotypes possessed the same ALS specific activities. Therefore, Hra represents a second independent mutation at or near the SuRB locus that reduces the sensitivity of tobacco ALS activity to inhibition by sulfonylurea herbicides.