Cotton, Gossypium hirsutum L., plants expressing Cry1Ac and Cry1F insecticidal crystal proteins of Bacillus thuringiensis Berliner (Bt) were evaluated against selected lepidopteran pests including fall armyworm, Spodoptera frugiperda (J. E. Smith), beet army worm, Spodoptera exigua (Hübner), and soybean looper, Pseudoplusia includens (Walker). Studies were conducted in a range of environments, challenging various cotton tissue types from several varieties containing a combination of Cry1Ac and Cry1F proteins. In fresh tissue bioassays of mature leaves and squares (flower buds) and in artificial field infestations of white flowers, plants containing Cry1Ac:Cry1F significantly reduced levels of damage (leaf defoliation, bract feeding, penetrated squares and bolls, and boll abscission) and induced significantly greater mortality (90–100%) of fall armyworm compared with that on non-Bt cotton plants. Plants containing Cry1Ac:Cry1F conferred high levels (100%) of soybean looper mortality and low levels (0.2%) of leaf defoliation compared with non-Bt cotton. Beet armyworm was relatively less sensitive to Cry1Ac:Cry1F cotton plants compared with fall armyworm and soybean looper. However, beet armyworm larval development was delayed 21 d after infestation (DAI), and ingestion of plant tissue was inhibited (14 and 21 DAI) on the Cry1Ac:Cry1F plants compared with that on non-Bt cotton plants. These results show Cry1Ac:Cry1F cotton varieties can be an effective component in a management program for these lepidopteran pest species. Differential susceptibility of fall armyworm, beet armyworm, and soybean looper larvae to Cry1Ac:Cry1F cotton reinforces the need to sample during plant development and respond with a foliar insecticide if local action thresholds are exceeded.