Seeds were collected from an imidazolinone-resistant (R) population of smooth pigweed near Marion, MD, and from an imidazolinone-susceptible (S) population near Painter, VA, and grown in the greenhouse. Acetolactate synthase (ALS) enzyme was extracted from both biotypes and assayed in the presence of CGA 152005, chlorimuron, halosulfuron, imazaquin, imazethapyr, nicosulfuron, primisulfuron, pyrithiobac, rimsulfuron, and thifensulfuron to determine if an altered ALS was the mechanism of resistance in the R biotype and to determine if this biotype was cross-resistant to other ALS inhibitor herbicides. The inhibitor concentration required to cause a 50% reduction in ALS activity (I50) was calculated for each herbicide. ALS from the R biotype was approximately 71-, 109,000-, and 9-fold more resistant to imazaquin, imazethapyr, and rimsulfuron, respectively, than that from the S biotype. ALS from the R biotype was approximately threefold more sensitive to pyrithiobac and thifensulfuron than that from the S biotype. R ALS was also slightly more tolerant to CGA 152005 and nicosulfuron and slightly more sensitive to primisulfuron and chlorimuron. ALS from both biotypes generally responded similarly to halosulfuron. Resistance in the R biotype was due to an altered form of ALS that is insensitive to the imidazolinone herbicides and rimsulfuron.