Differential response of three weed species to prosulfuron was examined through studies of acetolactate synthase (ALS) sensitivity, uptake, translocation, and metabolism of the herbicide. Sensitivity of common cocklebur, common lambsquarters, and sicklepod ALS to prosulfuron was similar with I50 values of 3.3, 4.5, and 8.8 nM, respectively. Absorption of the herbicide increased with time for all three weed species. Percent absorption 2 d after application for common cocklebur, common lambsquarters, and sicklepod was 8, 33, and 26%, respectively. An average of 53% of the absorbed herbicide remained in the treated leaves of common cocklebur, 80% in common lambsquarters, and 93% in sicklepod. Approximately 33, 15, and 5% of the absorbed herbicide was translocated into the upper shoots of common cocklebur, common lambsquarters, and sicklepod, respectively. Metabolism of 14C-prosulfuron 1, 2, and 4 d after application was greater in sicklepod than in common lambsquarters or common cocklebur. The results suggest a combination of differential rates of translocation and metabolism may account for the differing sensitivity of these weeds at the whole plant level.