Hypoxia-inducible factor (HIF) prolyl-hydroxylase domain-containing proteins (PHDs) are 2-oxoglutarate-dependent hydroxylases that regulate HIF turnover in the cell, thereby controlling the cellular response to hypoxic conditions. There are three PHD isoforms which are promising targets for treatment of heart attack, stroke, and other ischemic events. Current PHD inhibitors lack isoform specificity, thereby limiting their therapeutic potential. We are working toward structural characterization of all three isoforms to inform more effective development of isoform-specific inhibitors. Toward this goal, we have developed a method for high-yield recombinant production of the catalytic domain of PHD2 (trPHD2) and full-length PHD3 from inclusion bodies in E. coli. We have optimized refolding of the PHDs to minimize aggregation and preserve enzymatic activity. Activity is assessed by a fluorescence assay via derivatization of 2-oxoglutarate. These efforts provide the needed groundwork for future assessment of protein structures by NMR and subsequent isoform-selective drug design. This work was supported by a research grant from the New Jersey Health Foundation.