The methylotrophic yeast Pichia pastoris is used as a host system to produce recombinant proteins for a variety of applications, from food products to pharmaceuticals. However, despite the success of the P. pastoris expression system, relatively little is known about the molecular biology of the promoter utilized in this system, namely alcohol oxidase I (AOX1) promoter. In order to better utilize the AOX1 promoter, I propose to study regulation of the promoter using three approaches, 1) purify and characterize the regulatory proteins that bind to the AOX1 promoter and 2) overexpress his-tagged Methanol Expression Regulator 1 (Mrx1) protein to determine binding sites on the AOX1 promoter 3) determine the cis-acting regulatory sequences and design synthetic promoters. In addition, I will generate new host strains in which activator protein(s) will be over-expressed and expression of the repressor protein(s) will be suppressed. These studies will identify cis- and trans-acting factors that dictate the AOX1 promoter strength, in turn effect heterologous protein production. The elucidation of the AOX1 promoter control mechanism will benefit industrial and academic scientists who utilize P. pastoris expressions system in their studies.