Phenylketonuria is an autosomal recessive human genetic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene. In the present work we have used different expression systems to reveal folding defects of the PAH protein caused by phenylketonuria mutations L348V, S349L, and V388M. The amount of mutant proteins and/or the residual activity can be rescued by chaperonin co-overexpression in Escherichia coli or growth at low temperature in COS cells. Thermal stability profiles and degradation time courses of PAH expressed in E. coli show that the mutant proteins are less stable than the wild-type enzyme, also confirmed by pulse-chase experiments using a coupled in vitro transcription-translation system. Size exclusion chromatography shows altered oligomerization, partially corrected with chaperonins coexpression, except for the S349L mutant protein, which is recovered as inactive aggregates. PAH subunit interaction is affected in the S349L protein, as demonstrated in a mammalian two-hybrid assay. In conclusion, serine 349, located in the three-dimensional structure lining the active site and involved in the structural maintenance of the iron binding site, is essential for the structural stability and assembly and also for the catalytic properties of the PAH enzyme, whereas the L348V and V388M mutations affect the folding properties and stability of the protein. The experimental modulation of mutant residual activity provides a potential explanation for the existing inconsistencies in the genotype-phenotype correlations.