A shattercane biotype resistant to primisulfuron was identified during greenhouse evaluations of seed obtained from southeastern and south-central Nebraska fields previously treated with primisulfuron or nicosulfuron a minimum of 3 consecutive yr. Absorption, translocation, metabolism, and acetolactate synthase (ALS) assay experiments were conducted to determine resistance mechanism(s) by comparing ALS-susceptible forage sorghum (ROX) to resistant shattercane (RS). The ROX had 10 and 12% greater 14C absorption than RS 24 and 96 hours after treatment (HAT), respectively. Absorption of 14C increased over time for both ROX and RS, while 14C translocation from the treated leaf to the shoots and roots was similar for ROX and RS. Unmetabolized primisulfuron accounted for > 80% of the radioactivity recovered 24 h after application for both ROX and RS. The ROX and RS had similar ALS-specific activities and total protein concentrations. Km values for RS and ROX were 10.4 and 5.8 mM pyruvate, respectively. The ALS isolated from RS was less sensitive than ROX to inhibition by primisulfuron. The I50 values for RS and ROX were 231 and 0.025 μM primisulfuron, respectively. The mechanism of primisulfuron resistance in this RS biotype is an altered ALS with decreased sensitivity to primisulfuron.