An enzyme with 6-pyruvoyl tetrahydropterin (6PPH4) (2'-oxo)reductase activity was purified to near homogeneity from whole rat brains by a rapid method involving affinity chromatography on Cibacron blue F3Ga-agarose followed by high performance ion exchange chromatography and high performance gel filtration. The enzyme has a single subunit of Mr 37,000 and has a similar amino acid composition to previously described aldoketo reductases. The reductase activity is absolutely dependent on NADPH, will only catalyze the reduction of the C-2'-oxo group of 6PPH4, and is inactive towards the C-1'-oxo group. However, the enzyme also shows high activity towards nonspecific substrates, such as 4-nitrobenzaldehyde, phenanthrenequinone, and menadione. The role of this 6PPH4 reductase in the formation of tetrahydrobiopterin (BH4) was investigated. Measurements were made of the rate of conversion of 6PPH4, generated from dihydroneopterin triphosphate with purified 6PPH4 synthase, to BH4 in the presence of mixtures of pure sepiapterin reductase and the 6PPH4 (2'-oxo)reductase purified from rat brains. The results suggest that when sepiapterin reductase activity is limiting, a large proportion of BH4 synthesis proceeds through the 6-lactoyl intermediate. However, when sepiapterin reductase is not limiting, most of the BH4 is probably formed via reduction of the other mono-reduced intermediate which is produced from 6PPH4 by sepiapterin reductase alone.