The cotton bollworm, Helicoverpa zea (Boddie), is one of the most important insect pests of cotton, Gossypium hirsutum L. Cry1Ac protein expressed in transgenic Bt cotton has been widely used for control of cotton bollworms since 1996. However, extensive and constant exposure of cotton bollworms to this protein may result in the development of resistance. To estimate the susceptibility of the field populations of cotton bollworm to this Bt protein, we compared the survivorship of F1 or F2 generations of field-derived cotton bollworms collected from Mid-south regions in the U.S. to Cry1Ac protein with that of susceptible counterparts using diet incorporated and cotton leaf tissue bioassays. In the leaf tissue tests, the 7-day survivorship of all field collected populations (54.7-66.1%) was significantly higher than that of susceptible strain (26.1%) on the leaf tissues of Cry1Ac-only cotton. The LC50 of the susceptible strain to Cry1Ac protein was 0.265 µg/g with a 95% confidence interval of 0.207-0.339 µg/g. However, dose-response bioassays showed all the field collected populations except for one from Louisiana showed a significant resistance level (> 37.7-fold) to Cry1Ac protein with the LC50 > 10 µg/g, compared to the susceptible strain. These results indicated that the susceptibility of cotton bollworm to Cry1Ac protein has been decreased in the fields in the Mid-south regions of U.S.