Phenylketonuria (PKU) is a rare inborn error of metabolism. To avoid severe complications of PKU, including serious brain damage, patients need to follow a very strict diet for their entire life. Since 2008, some PKU patients are treated with a drug called tetrahydrobiopterin (BH4) which allows them to follow a less strict diet or sometimes no diet at all. This thesis evaluated the effects and difficulties of BH4 treatment in PKU patients. First, we focussed on the (long-term) effects of BH4 treatment, including nutritional status, growth and quality of life. We found that there was little evidence for a positive effect of BH4 on these outcomes, which was especially surprising for quality of life. Second, since not all PKU patients respond to BH4 treatment, we assessed different ways to test and define BH4 responsiveness. Our results indicated that methods to assess responsiveness need further optimization, and findings from a large international practice survey indicated little consensus on the topic of defining BH4 responsiveness. Third, we focussed on the effects of BH4 on the brain. Although there are many publications suggesting possible positive effects of BH4 on cerebral functioning, our study in embryonic mice neurons indicates that we should also look at possible toxic effects of BH4. Overall, while BH4 treatment can be considered as a major step forward in the treatment of PKU, there are still many challenges that need be overcome to more effectively implement this relatively new therapeutic option.