The insertion of genes encoding insecticidal Cry1A δ-endotoxins from the bacterium Bacillus thuringiensis Berliner variety kurstaki (Bt) into varieties of cotton (Gossypium spp.) was undertaken to assist in the control of a range of lepidopteran pests. In Australia, where Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) are major pests, the level of control is useful, but not complete, because efficacy declines as the crop matures. Fluctuations in the efficacy of Bt cotton, to the extent that some insects survive, provide opportunities for H. armigera to develop resistance to the Bt toxin. Therefore, variations in the efficacy of Bt cotton need to be understood if we are to plan rational resistance management strategies to retard the rate of the development of resistance. We measured the changes in efficacy associated with plant development over the growing season, in the field and glasshouse. In addition, the levels of Cry1Ac protein toxin and cry1Ac RNA were determined. In this first demonstration of the relationship between these three factors, we found that the developmental decline in bioefficacy in field-grown plants was associated with reduced cry1Ac transcript levels and Bt toxin levels in postsquaring cotton. In addition, changes in plant chemistry associated with the maturation of the cotton plant were observed to contribute to changes in the efficacy of Bt toxin. Results from the field and glasshouse suggested that variations in efficacy within the growing season and between seasons also may be influenced by environmental factors.