WideStrike® Acala cotton is a two-gene, in-plant trait that provides broad-spectrum and season-long control of lepidopteran insect pests, and the varieties available in California also have resistance to glyphosate. There have been indications that WideStrike cotton has some glufosinate tolerance as well, so the level of tolerance to glufosinate needed to be ascertained. A 2-yr (2008 and 2009) study was conducted in California to evaluate the potential crop injury caused by three different rates (0.59, 0.88, and 1.76 kg ai ha−1) of glufosinate–ammonium at four different growth stages (cotyledon, 2-node, 5- to 6-node, and 18- to 19-node stages) of WideStrike Acala cotton. The effects of these treatments on the cotton plants and yield were closely monitored. Glyphosate at 1.54 kg ae ha−1 was applied at all cotton growth stages as a standard application, and a nontreated control was included. The greatest level of injury (58%) was observed with the highest rate of glufosinate applied at both the cotyledon and the two-node stage of cotton. However, injury was less than 10% following glufosinate at 0.59 kg ha−1 applied at the 18- to 19-node stage. The level of injury increased with the higher application rate of glufosinate at all crop growth stages. In 2008 and 2009, the glufosinate treatments had no effect on cotton lint yield. Therefore, the study showed that glufosinate can be applied safely topically at 0.59 kg ha−1 at the cotyledon- to 2-node stage or as POST-directed spray between the 5- to 19-node stages. Although injury occurred at this rate, the plants recovered within 2 to 3 wk of the treatment. Increasing glufosinate rates beyond 0.59 kg ha−1 can increase the possibility of greater crop injury.