A 4 field site study was performed in which Bollgard II (containing 2 lepidopteran active Bt proteins: Cry1Ac and CryX) and DP50B (original Bollgard^® containing only Cry1Ac) cotton tissue samples were collected throughout the growing season and evaluated for total lepidopteran bioactivity using a sensitive Heliothis virescens quantitative bioassay which utilized purified Cry1Ac as the quantitative standard. In addition, protein-specific ELISA assays were performed upon the same tissue samples to determine relative levels of both insect control proteins. Total lepidopteran bioactivity, expressed in Cry1Ac equivalents, was greatly increased in Bollgard II tissue samples. Overall means were four times as great for Bollgard II as for DP50B (the "parent" Bollgard^® variety); overall means were 3 times as great for terminal foliage and 6 times as great for square tissue. This relative increase in lepidopteran activity was observed at every sampling time (from 4-leaf stage to 6 weeks after first bloom) and at every field site. ELISA evaluations showed that the presence of the second protein (CryX) had no deleterious effect on the levels of the first Bollgard^® protein (Cry1Ac) as measured in DP50B. Also, relative levels of the two Bt proteins remained relatively constant over time and across field sites. A main-effect ANOVA determined that, in addition to the Bollgard II-Bollgard^® difference, field site, sampling time, and plant tissue type were all significant sources of variability among levels of lepidopteran bioactivity; although the tissue type variability was due solely to differences between terminals and squares within DP50B; when evaluated alone, there was no statistical difference in the lepidopteran activity between Bollgard II squares and terminals. These data strongly suggest that the greatest single effect of the addition of the CryX protein to Bollgard^® to produce Bollgard II is likely to be greatly increased lepidopteran activity, especially in reproductive tissues.