In addition to its well-known cofactor roles for aromatic amino-acid hydroxylases, an essential role of tetrahydrobiopterin (BH 4 ) for nitric oxide synthases (NOS) has been recently established ( 1 – 3 ). All three isoforms of NOS contain high-affinity binding sites for BH 4 . This cofactor is enzymatically synthesized in the cell from guanosine 5′-triphosphate (GTP) by the sequential actions of three distinct enzymes ( see Chapter 24 , Fig. 1 , this volume). The first and rate-limiting enzyme in the biosynthetic pathway of BH 4 is GTP cyclohydrolase I (EC 3.5.4.16). Cellular expression of this enzyme is subject to varied and multi-level control that is cell-type specific. For example, the activity of GTP cyclohydrolase I in immune cells is normally either low or undetectable, but it is coinduced with the inducible NOS (NOS 2) in response to immunoreactive cytokines and bacterial lipopolysaccaride ( 4 – 6 ). Hepatic GTP cyclohydrolase I is constitutively expressed and is regulated by BH 4 and phenylalanine through interaction with a specific GTP cyclohydrolase I feedback regulatory protein (GFRP) ( 7 ). Although the role of this regulator protein in nonhepatic tissues is unclear, an interesting possibility is that it may be involved in regulating the production of NO by NOS. On the other hand, the two enzymes catalyzing the subsequent steps to BH 4 are constitutively expressed in cells and there is little evidence suggesting regulation of the activities of these enzymes.   Fig. 1.  A chromatographic profile of neopterin. Isolation and cell culture of rat hepatocytes were performed as described (20). Hepatocytes (5 � 10 6 ) were plated onto a 90-mm gelatin-coated Petrie dish and incubated for 2 h at 37�C. Crude extracts were obtained as described in the Appendix. Two-hundred microliters of the crude extracts was passed through a NICK spin column. Fifty microliters of the eluate was used for GTP cyclohydrolase I assay. After performing the enzyme assay as described in the Methods Section, 30 �L from 850 �L of the supernatant obtained at the step 9 was injected into a Partisil 10 ODS reversed-phase column. Peak at 7.37 min, neopterin.