Folate, also known as vitamin B9, is an essential dietary vitamin that provides the donor group for one carbon transfer reactions. Deficiency in folate is associated with neural tube birth defects (NTDs), cancer, cardiovascular disease, and anemia. In the US enriched food products including bread, pasta, and cereal are fortified with folic acid, the synthetic analog of folate. While effective in reducing NTDs, this practice is costly and not economically practical in developing countries. Folate biofortification, increasing the natural folate level in foods by metabolic engineering, has been proposed as a sustainable alternative to food fortification with folic acid. To increase folate levels in peanut seed, GTP cyclohydrolase I from Arabidopsis thaliana (AtGCHI) was introduced into peanut by biolistic transformation. Plant transformation vectors were constructed using publicly available or licensable vector components to avoid intellectual property restrictions that hinder commercialization. Thirteen peanut cultivars were evaluated for transformation efficiencies and regeneration potential. Expression levels of the AtGCHI transgene were determined by quantitative real-time PCR. The endogenous peanut GCHI (AhGCHI) was isolated and sequenced. Studies were conducted to test whether heterologous over-expression of AtGCHI altered expression of the endogenous AhGCHI. Seed-specific expression of AtGCHI does not affect AhGCHI transcript accumulation. For validation of the proposed folate biofortification strategy, vitamin quantification will be required. A liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed to identify and quantify the different forms of folate. However, additional work will be needed to determine sensitivity of the instrument, to optimize vitamin extraction, and to increase sufficient seed for vitamin extraction and analysis. Peanut products derived from folate biofortified peanut kernels will have a niche market in the United States, but there is a larger global implication as a mechanism for sustainable delivery of essential vitamins to populations that can not adopt synthetic vitamin supplementation/fortification. Successful demonstration of increased folate in peanut will result in better vitamin availability for populationssonsuming peanut based foods as a dietary staple. / Ph. D.