Two biotypes of Scirpus mucronatus not controlled with the herbicide bensulfuron-methyl in rice fields were characterized by using field, greenhouse, and laboratory techniques. Seeds were collected in two rice areas [Parral (R1) and Linares (R2)], where bensulfuron-methyl at 150 g ha(-1) did not control S. mucronatus. A third seed sample of S. mucronatus susceptible (S) to bensulfuron-methyl was collected in an area from Chile. The dose-response studies confirmed resistance to bensulfuron-methyl in R1 and R2 S. mucronatus biotypes; ratios (R/S) of the ED(50) values of resistant to susceptible plants were 1719 and 1627 for R1 and R2, respectively. The biotype R1 also showed strong cross-resistance (ratios ranging from 1719 to 43) to sulfonylureas (bensulfuron-methyl, cyclosulfamuron, ethoxysulfuron, imazosulfuron, and pyrazosulfuron-ethyl) and imidazolinone (imazamox) and a weak cross-resistance (ratio of 1.705) to pyrimidinyloxybenzoates (bispyribac-sodium), all ALS inhibiting herbicides used in rice. Absorption, translocation, and metabolism results did not explain the differences in susceptibility among biotypes. The in vitro assays confirmed cross-resistance to all ALS inhibitors tested and the level of cross resistance was bensulfuron-methyl > imazosulfuron ≫ cyclosulfamuron ≫ pyrazosulfuron-ethyl ≫ ethoxysulfuron > imazamox ≫ bispiribac-sodium. Molecular studies demonstrated that the Pro197His amino acid substitution on the ALS enzyme could explain the loss of affinity for the ALS-inhibiting herbicides.