We have used three complementary in vitro systems to express the human phenylalanine hydroxylase (PAH) gene at high levels. Recombinant PAH was expressed in Escherichia coli (as a fusion protein), in human kidney cells and in a cell-free in vitro transcription-translation system. These systems were used to characterize a novel kinetic variant form (D143G) of the enzyme. The recombinant D143G mutant enzyme had the same physicochemical properties as the wild-type PAH and was stable when expressed in eukaryotic cells. Enzyme activity studies of the D143G mutant enzyme, produced in the three expression systems, revealed a kinetic variant form with reduced affinity for L-Phe (about 2.4-fold increase in the S0.5 value) as well as reduced affinity for tetrahydrobiopterin (BH4) (about 2-fold increase in the apparent Km). At standard assay conditions (1 mM L-Phe, t5 microM BH4) the residual activity of the mutant enzyme was high and variable (52%, 33%, and 102%) when analysed in the three different systems. The high residual activities of the mutant enzyme obtained at these conditions were not in agreement with the classical PKU phenotype found in a patient compound heterozygous for the termination mutation G272X and the novel D143G mutation. However, when the D143G mutant enzyme was assayed at lower concentrations of L-Phe (100-300 microM) and BH4 (10 microM) the residual activities were compatible with severely reduced hydroxylation of L-Phe and the classical PKU phenotype.