A subtype of phenylalanine hydroxylase (PAH) deficiency that responds to cofactor (tetrahydrobiopterin, BH₄) supplementation has been associated with phenylketonuria (PKU) mutations. The underlying molecular mechanism of this responsiveness is as yet unknown and requires a detailed in vitro expression analysis of the associated mutations. With this aim, we optimized the analysis of the kinetic and cofactor binding properties in recombinant human PAH and in seven mild PKU mutations, i.e., c.194T>C (p.I65T), c.204A>T (p.R68S), c.731C>T (p.P244L), c.782G>A (p.R261Q), c.926C>T (p.A309V), c.1162G>A (p.V388M), and c.1162G>A (p.Y414C) expressed in E. coli. For p.I65T, p.R68S, and p.R261Q, we could in addition study the equilibrium binding of BH₄ to the tetrameric forms by isothermal titration calorimetry (ITC). All the mutations resulted in catalytic defects, and p.I65T, p.R68S, p.P244L, and most probably p.A309V, showed reduced binding affinity for BH₄. The possible stabilizing effect of the cofactor was explored using a cell‐free in vitro synthesis assay combined with pulse‐chase methodology. BH₄ prevents the degradation of the proteins of folding variants p.A309V, p.V388M, and p.Y414C, acting as a chemical chaperone. In addition, for wild‐type PAH and all mild PKU mutants analyzed in this study, BH₄ increases the PAH activity of the synthesized protein and protects from the rapid inactivation observed in vitro. Catalase and superoxide dismutase partially mimic this protection. All together, our results indicate that the response to BH₄ substitution therapy by PKU mutations may have a multifactorial basis. Both effects of BH₄ on PAH, i.e., the chemical chaperone effect preventing protein misfolding and the protection from inactivation, may be relevant mechanisms of the responsive phenotype. Hum Mutat 24:388–399, 2004. © 2004 Wiley‐Liss, Inc.