The molecular basis and mode of inheritance of resistance to ACCase-inhibiting herbicides were investigated in a biotype of Lolium rigidum Gaud that has been discovered in Israel. Dose-response experiments at the whole-plant level have revealed that the resistant biotype was more resistant (6.3- to 40-fold) than the susceptible wild-type to ACCase-inhibiting herbicides. A 276-bp genomic DNA encoding the carboxyltransferase domain within the chloroplastic ACCase from resistant and susceptible biotypes were amplified by PCR and analyzed. Sequence comparison revealed that a single isoleucine-to-leucine substitution differentiated ACCases from susceptible and resistant biotypes (corresponds to residue 1769 of wheat ACCase, Acc No AF029895). A PCR amplification of specific alleles (PASA) method was developed to detect the allele composition leading to isoleucine-leucine mutation. ACCase extracted from homozygote resistant, heterozygote and homozygote susceptible plants showed IC50 values of 25.8, 5.6 and 0.6 microM, respectively, suggesting that alteration in the ACCase is governed by a co-dominant gene. The inheritance studies confirmed that the resistance of L rigidum to ACCase-inhibiting herbicides is governed by a single, nuclear and co-dominant gene.