The Quantase R Phenylalanine Assay, an enzymatic/colorimetric method for the determination of phenylalanine (Phe) in dried bloodspots, was evaluated for the neonatal screening on PKU. The within run variation (N=12) for bloodspot samples with 0.15, 0.29 and 0.53 mmol/l was 10%, 5% and 5%, respectively. The between run variation (N=56) for bloodspot samples with 0.38 and 0.96 mmol/l was 9% and 8%, respectively. The mean concentration and standarddeviation in 33630 bloodspot samples was 0.077 +- 0.025 mmol/l (%CV=32%). The measured Phe-concentration was elevated (>=0.24 mmol/l) in 13 samples (0.039%). Reanalysis of these samples, in the same bloodspots, revealed a Phe-concentration of <0.12 mmol/l in 6 samples. These 6 samples were also found to be 'negative' in the Guthrie-test. Therefore, 6 samples (0.018%) were 'false-elevated'. For 5 of these samples no explanation was found. In 5770 bloodspot samples Guthrie test. All 'negative' samples in the Quantase were also 'negative' in the Guthrie. According to the manufacturer the absorbance has to be read at 570 nm. To reduce the number of samples with a 'false-elevated' concentration, we recommend to read the absorbances both at 570 nm and at 690 nm to correct for non-specific absorbance (dual wavelength). We used the Millipore Multiscreen Assay System for the extraction of the phenylalanine from the bloodspots. This system appeared to function well in practive. The special filterplates are rather expensive, but under stringent conditions the plates can be recycled three times. A visual control on the correct transfer of the extracts is necessary.