Our objective was to study the influence of pegylated interferon-α2b (PEG-IFN-α) on the metabolism of amino acids and pteridines. We used an exploratory study into plasma concentrations of large neutral amino acids, 5-hydroxyindolacetic acid (5-HIAA), total biopterin (BIOP) and neopterin (NEOP) in 40 high-risk melanoma patients. Patients were randomized to treatment with PEG-IFN-α once a week in a dose of 6 μg/kg/week s.c. during 8 weeks, followed by a maintenance treatment of 3 μg/kg/week s.c. or to observation only. We found that treatment with PEG-IFN-α decreases tryptophan (TRP) concentrations in the first 3 months of treatment to a maximum of 25.3% compared to controls [95% confidence interval (CI): 14.9 to 34.4]. The TRP:LNAA ratio, an index for the availability of TRP to the central nervous system (CNS), decreases during 6 months with 18.8% (95% CI: 11.9 to 25.2). Concentrations of NEOP rose; however, concentrations of BIOP, the sum of tetrahydrobiopterin [BH(4)] and its oxidative products, did not decrease. The ratio of phenylalanine to tyrosine was increased with 11.7% (95% CI: 1.0 to 23.5) during 6 months. We conclude that, like conventional IFN-α, PEG-IFN-α lowers TRP concentrations and decreases the availability of TRP to the CNS. PEG-IFN-α has a similar influence on pteridine metabolism as standard IFN-α. If a lowered availability of TRP and a consequent decrease of serotonergic neurotransmission are indeed a mechanism underlying neuropsychiatric side-effects of IFN-α, patients on PEG-IFN-α are not at a lower risk of developing neuropsychiatric side-effects as patients on conventional IFN-α.