Transgenic Bt-cotton expresses insecticidal Cry proteins via Cry genes, introduced from the soil bacterium Bacillus thuringiensis (Bt). Data on levels of Cry proteins expressed in transgenic Bt-crops is important for assessing levels of environmental exposure. During investigations into the rhizosphere impacts of Cry proteins from GM cotton we found that Cry1Ac expression throughout the season was significantly lower in roots of glasshouse-reared cotton plants (average of 0.03 g/g in the roots) compared to the same Bt-cotton varieties grown in the field (0.14 g/g in the roots). We subsequently undertook a whole plant field assessment of both Cry1Ac and Cry2Ab expression in stem, leaves, root, and flowers, squares and bolls of Australian cotton varieties Sicot 289 Bollgard ® II (289B) and Sicot 289 Bollgard ® II Roundup Ready (289BR) over an entire season. Significant differences in the expression levels of Cry1Ac versus Cry2Ab occurred in the whole plant throughout the season. Cry1Ac levels remained relatively constant at an average of 6.1 g/g whilst Cry2Ab levels averaged 29.0 g/g, but decreased over time. Analysis of whole plant expression levels, plant stand densities, and aspects of crop management, estimated that levels of Cry1Ac and Cry2Ab deposited in the soil at the end of the season were 0.26 and 0.16 g/g soil, respectively. The undertaken experiments highlight that assessments of environmental loading of proteins from GM plants would be improved with the use of field grown plants, whole plant assessments, increased knowledge on fate and persistence of GM proteins in the soil, and refinement of current ELISA methodologies.