Assessment of total neopterin and tetrahydrobioterin (BH4) concentrations in cerebrospinal fluid (CSF) can be used to identify potential disorders of BH4 biosynthesis. In this study, we demonstrate that exposure of CSF to nitric oxide leads to an accelerated degradation of BH4 but does not affect the total neopterin concentration. These data suggest that in those conditions associated with increased nitric oxide formation, perturbation of the total neopterin to BH4 ratio could occur. Under such circumstances a putative diagnosis of a defect in BH4 biosynthesis may erroneously be proposed. Assessment of central nitric oxide generation may therefore be a useful adjunct to the determination of CSF pterin status.