New trait technology incorporating 2,4-D resistance in soybean is dependent upon the ability of the plant to metabolize 2,4-D by the aryloxyalkanoate dioxygenase-12 protein (AAD-12). Our objectives were to determine AAD-12 expression during the daytime, throughout the leaf canopy, and before and after 2,4-D treatment for the events DAS-68416-4 and DAS-21606-3. Field experiments were conducted near Wanatah, IN in 2009 and Fowler, IN in 2009, 2010, and 2011. During the daytime, total AAD-12 expression was lowest between 12:30 and 15:30, averaging 161 ng cm−2, as compared to an average of 245 ng cm−2 in the morning and 243 ng cm−2 in the evening. The youngest fully emerged trifoliate in the DAS-68416-4 event had the highest AAD-12 expression, with means ranging from 369 to 390 ng cm−2, while the older leaves maintained a lower level of expression, 171 to 211 ng cm−2. The youngest leaves of event DAS-21606-3 had the highest level of AAD-12 expression (205 to 225 ng cm−2), while the level of AAD-12 was lower in older leaves (71 to 149 ng cm−2). In general, 2,4-D treatments did not reduce AAD-12 expression at 3, 7, 14, and 21 days after treatment; however, in a few instances AAD-12 expression was increased or decreased by 8 to 11% after 2,4-D treatment. Expression of AAD-12 was between 152 to 390 ng cm−2 for DAS-68416-4 and from 71 to 244 ng cm−2 for DAS-21606-3.