Soil samples were collected from within and outside six fields where insect-resistant transgenic cotton (Bollgard) encoding the Bacillus thuringiensis Berliner (Bt) subsp. kurstaki cry1Ac gene had been grown and subsequently incorporated into soil by postharvest tillage for 3–6 consecutive years. The level of Cry1Ac protein in these samples (collected 3 mo after the last season’s tillage) was evaluated using both enzyme-linked immunosorbent assays (ELISA) and bioassays with a susceptible insect species, Heliothis virescens (F.), the tobacco budworm. Both methods revealed that no detectable Cry1Ac protein was present in any of the soil samples collected from within or outside the Bollgard fields. Based on the results from reference standards, the limit of detection for the ELISA was 3.68 ng of extractable protein per gram of soil, and that of the bioassay (measured by EC50) was 8 ng of biologically active protein per gram of soil. Together, these findings demonstrate that the amount of Cry1Ac protein accumulated as a result of continuous use of transgenic Bt cotton, and subsequent incorporation of plant residues into the soil by postharvest tillage, is extremely low and does not result in detectable biological activity.