Prokaryotic fatty acid biosynthesis is an important process to study due to its necessity for cell survival and production of highly reduced carbon chains. A characterization of several proteins involved in E. coli fatty acid biosynthesis is presented. This work provides a method for generating large amounts of pure malonyl acyl-carrier-protein using both the wild type malonyl-CoA:holo-ACP transacylase (FabD) and a variant studied further in our lab. This preparation overcomes the phenomena of a 50:50 mixture of malonyl and acetyl-ACP in the reaction and is crucial for the efficacy of future assays involving malonyl-ACP. A new assay has been developed to measure activity of the FabD enzyme using a fluorescent probe and can be scaled from a rapid to high throughput assay using 96 well plates. FabD variants were studied with the above assay and kinetic constants were determined for several different CoA substrates with enhanced chemical functionality. The structure of holo-ACP synthase in apo and acyl-carrier-protein bound forms was investigated in depth using a combination of X-ray crystallography, NMR spectroscopy, and isothermal titration calorimetry. This work is a comprehensive study of the enzymes involved in the first steps of fatty acid biosynthesis using several biochemical and biophysical techniques.