Technical Abstract: Ultra-low gossypol cottonseed (ULGCS) lines were developed using RNAi-knockdown of d-cadinene synthase gene(s) in Gossypium hirsutum. This RNAi-mediated ULGCS trait has exhibited multi-generational stability under greenhouse conditions. The purpose of the current study was to assess the stability and specificity of the ULGCS trait in the transgenic lines under field conditions. In addition, we also investigated whether the engineered trait had any adverse effects on the yield/quality of the seeds or fiber. Trials were conducted over a period of three years and the results show that the ULGCS trait was stable under field conditions and the non-seed tissues contained normal, wild-type levels of gossypol and related terpenoids. The transgenic plants were no more vulnerable to diseases or pests compared to their non-transgenic counterparts. We did not observe any negative effects on either the yield or quality of the fiber in the transgenic lines. In fact, in two of the three years, transformants had higher yield and better fiber quality. A similar trend was observed for seed yields in the transgenic lines. Compositional analysis was performed on the seeds obtained from plants grown in the field during 2009. Overall, the composition of ULGCS was similar to that of wild-type cottonseeds, with the exception of oil content. Interestingly, a follow up study involving NMR-based oil content analysis on seeds obtained from all three years confirmed that the ULGCS had significantly higher (4-8%) oil content compared to the seeds from the parental line. Field trial results confirmed the stability and specificity of the ULGCS trait suggesting that this RNAi-based product has the potential to be commercially viable. Thus, it may yet be possible to enhance and expand the nutritional utility of the enormous, annual cottonseed output to fulfill the ever-increasing needs of humanity.