AbstractEnlist E3^TM soybean cultivars permit over-the-top application of labeled glyphosate, glufosinate, and 2,4-D choline products. Increased Enlist E3^TM trait adoption and use of 2,4-D choline postemergence across U.S. soybean production systems raise concerns regarding potential for 2,4-D off-target movement (OTM). A large-scale drift experiment was established near Sun Prairie, WI, and Arlington, WI, in 2019 and 2020, respectively. A 2,4-D-resistant soybean cultivar was planted in the center of the field (∼3 ha), while the surrounding area was planted with a 2,4-D-susceptible cultivar. An application of 785 ae ha⁻¹ 2,4-D choline plus 834 g ae ha⁻¹ glyphosate was completed within the center block at R2 and V6 growth stages on August 1, 2019, and July 3, 2020, respectively. Filter papers were placed in-swath and outside of the treated area in one upwind transect and three downwind transects to estimate particle deposition. Low-volume air samplers ran for the 0.5-h to 48-h period following application to estimate 2,4-D air concentration. Injury to 2,4-D-susceptible soybean was assessed 21 d after treatment (0% to 100% injury). The 2,4-D deposition in-swath was 9,966 and 5,727 ng cm⁻² in 2019 and 2020, respectively. Three-parameter log-logistic models estimated the distance to 90% reduction in 2,4-D deposition (D₉₀) to be 0.63 m and 0.90 m in 2019 and 2020, respectively. In 2020, the 2,4-D air concentration detected was lower for the upwind (0.395 ng m⁻³) than the downwind direction (1.34 ng m⁻³), although both were lower than the amount detected in-swath (4.01 ng m⁻³). No soybean injury was observed in the downwind or upwind directions. Our results suggest that 2,4-D choline applications following label recommendations pose little risk to 2,4-D-susceptible soybean cultivars; however, further work is needed to understand 2,4-D choline OTM under different environmental conditions and the presence of other susceptible crops.