Tetrahydrobiopterin (BH4) belongs to a group of molecules known as pteridines and has numerous cellular functions. Most notably, it is an obligate cofactor for several important enzymes including aromatic amino acid hydroxylases and nitric oxide synthases (NOSs). In recent years, BH4?s role in NOS uncoupling has become one center of attention in this field of research. BH4 deficiency can lead to NOS uncoupling which is associated with cardiovascular pathologies. The levels of BH4 are determined by the activity of GTP cyclohydrolase (GTPCH), the key enzyme in BH4 biosynthesis. BH4 bioavailability is also influenced by the redox state of the cell as BH4 reacts with oxidants, forming cofactor inactive dihydrobiopterin (BH2). BH2 can be reduced to BH4 by dihydrofolate reductase (DHFR).Research at our institute showed that intracellular levels of BH4 in cultured endothelial cells are affected by growth media. Culturing cells in M199 and MCDB leads to diminished BH4 levels compared to cells cultured in DMEM. We investigated biosynthesis and recycling of BH4 in porcine aortic endothelial cells treated with different media by monitoring the enzyme activity of GTPCH and DHFR. DHFR activity did not differ significantly in cells treated with DMEM, M199 and MCDB and thus can be ruled out as reason for decreased BH4 levels. Before GTPCH activity could be measured, a HPLC enzyme assay had to be established. Several adjustments were made to a provided working protocol. Those changes included substituting the ascorbic acid used as a reducing agent by dithiothreitol (DTT). Incubating ascorbic acid with iodine oxidizing reagent in an alkaline range at 37 C caused a disturbing peak in the chromatogram which was prevented by using DTT. When GTPCH activity was measured, a trend towards reduced GTPCH activity in PAEC treated with M199 and MCDB could be shown, but further investigation is needed to confirm this tendency.