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Glasshouse and laboratory experiments were conducted on acetolactate synthase (ALS) homozygous resistant Solanum ptycanthum biotypes from Illinois (IL-R) and Indiana (IN-R), and homozygous susceptible biotypes from Illinois (IL-S) and Indiana (IN-S). Genetic similarity of biotypes was assessed by random amplified polymorphic DNA (RAPD) markers, which determined that the Illinois biotypes are more similar to each other than to the IN-R biotype. ALS enzyme activity from the IL-R and IN-R biotypes had I50 values of 362 and 352 μM imazamox respectively. Dose–response experiments using three- to four-leaf-stage plants of the IL-R and IN-R biotypes had GR50 values of 242 and 69 g ae ha−1 imazamox respectively. Whole-plant and ALS enzyme results are different than previously reported values in the literature, which was attributed in the current study to the original IN-R population having individuals that were segregating for ALS resistance. Metabolism studies showed no difference in percentage [14C]imazamox remaining between the IL-R and IN-R biotypes up to 72 h after treatment. The IL-S biotype metabolised [14C]imazamox approximately two times faster than the IL-R and IN-R biotypes and this trait was heritable. Response of F3 plants containing homozygous ALS-resistant alleles from the IL-R biotype in a genetic background of 50% Illinois and 50% Indiana biotypes suggests that genetic factors other than an altered target site or metabolism may also contribute to the magnitude of resistance at the whole-plant level in resistant biotypes. [ABSTRACT FROM AUTHOR]