The amount of Cry1Ac δ-endotoxin in transgenic Bacillus thuringiensis Berliner (Bt) or Bollgard cotton varies among commercial cultivars. These differences in expression have been correlated with survival levels in Lepidoptera, indicating that all Bollgard cultivars do not provide the same level of control. The objective of this study was to determine if differences in overall expression among commercial cultivars of Bollgard cotton were under simple genetic control. These findings could influence the way breeders select cultivars by evaluating for efficacy in insect control in addition to agronomic traits. Two sets of crosses were made in the greenhouse with cultivars that express the endotoxin at high and low levels. The parents and F1 and F2 generations were planted in the field. The amount of Cry1Ac was quantified using a commercial ELISA kit. Variances within the two F2 breeding populations were highly significant because of genetic segregation for Cry1Ac expression. Using the modified Castle-Wright formula, the estimation of the number of contributing genes in both breeding populations was small. These data show that genetic background has a major effect on Cry1Ac expression. Because backcrossing is the primary method used by commercial cotton breeders, the selection and use of donor and/or recurrent parents that will result in a high level of Cry1Ac expression is crucial.