Phenylketonuria (PKU) is an autosomal recessive disorder resulting in neurological and intellectual disability when untreated. However, even in treated patients there may be residual neurological impairment such as tremor. It has been suggested that the hyperphenylalaninaemia in patients with PKU reduces complex I (NADH:ubiquinone reductase) activity of the mitochondrial respiratory chain (MRC) and/or biosynthesis of coenzyme Q(10) (CoQ(10)), which acts as an electron carrier in the MRC, leading to impaired energy metabolism in the brain of patients with PKU and hence the neurological pathology. The aim of this study was to elucidate the mechanism of phenylalanine (Phe) toxicity on the MRC. We compared mean plasma and blood-spot Phe and mononuclear CoQ(10) levels in 17 patients with PKU and a tremor compared to 22 patients without tremor. Human 1321N1 astrocytoma cells were exposed to hyperphenylalaninaemia by the addition of 300 or 900 micromol/L of Phe to the cell culture medium. Following 96 h of culture we measured complex I and citrate synthase activities and CoQ(10) level. Results showed no significant difference in Phe or CoQ(10) levels in patients with tremor compared to those without tremor. Further, hyperphenylalaninaemia did not cause a significant reduction in complex I activity or CoQ(10) biosynthesis, even when taking into account the mitochondrial enrichment of the cell samples by expressing complex I and CoQ(10) as a ratio to citrate synthase. In conclusion, the results of this study suggest that hyperphenylalaninaemia does not contribute to the pathophysiology of PKU by causing a decrease in MRC complex I activity and/or CoQ(10) biosynthesis.