Experiments were conducted to evaluate a biotype of smooth pigweed that had survived applications of sulfonylurea (SU) and imidazolinone (IMI) herbicides in a single season. The source field had a history of repeated acetolactate synthase (ALS)-inhibiting herbicide use over several years. Whole-plant response experiments evaluated the resistant (R11) biotype and an ALS-inhibitor susceptible (S) smooth pigweed biotype to herbicides from the SU, IMI, pyrimidinylthiobenzoate (PTB), and triazolopyrimidine sulfonanilide (TP) chemical families. The R11 biotype exhibited 60- to 3,200-fold resistance to all four ALS-Inhibiting herbicide chemistries compared with the S biotype. Nucleotide sequence comparison of ALS genes from R11 and S biotypes revealed a single nucleotide difference that resulted in R11 having an amino acid substitution of aspartate to glutamate at position 376, as numbered relative to the protein sequence of mouseearcress. This is the first report of an amino acid substitution at this position of an ALS gene isolated from a field-selected weed biotype. To verify the role of this mutation in herbicide resistance, the ALS gene was cloned and expressed in Arabidopsis. Transgenic Arabidopsis expressing this ALS gene exhibited resistance to SU, IMI, PTB, TP, and sulfonylaminocarbonyltriazolinone ALS-Inhibiting herbicide classes.