In order to increase the tissue level of tetrahydrobiopterin (BH₄), supplementation with 6R-tetrahydrobiopterin (6RBH₄) has been widely employed. In this work, the effectiveness of 6RBH₄ was compared with 7,8-dihydrobiopterin (7,8BH₂) and sepiapterin by administration to mice. Administration of 6RBH₄ was the least effective in elevating tissue BH₄ levels in mice while sepiapterin was the best. In all three cases, a dihydrobiopterin surge appeared in the blood. The appearance of the dihydrobiopterin surge after BH₄ treatment suggested that systemic oxidation of the administered BH₄ had occurred before accumulation of BH₄ in the tissues. This idea was supported by the following evidences: 1) An increase in tissue BH₄ was effectively inhibited by methotrexate, an inhibitor of dihydrofolate reductase which reduces 7,8BH₂ to BH₄. 2) When the unnatural diastereomer 6SBH₄ was administered to mice, a large proportion of the recovered BH₄ was in the form of the 6R-diastereomer, suggesting that this BH₄ was the product of a dihydrofolate reductase process by which 7,8BH₂ converts to 6RBH₄. These results indicated that the exogenous BH₄ was oxidized and the resultant 7,8BH₂ circulated through the tissues, and then it was incorporated by various other tissues and organs through a pathway shared by the exogenous sepiapterin and 7,8BH₂ in their uptake. It was demonstrated that maintaining endogenous tetrahydrobiopterin in tissues under ordinary conditions was also largely dependent on an methotrexate-sensitive process, suggesting that cellular tetrahydrobiopterin was maintained both by de novo synthesis and by salvage of extracellular dihydrobiopterin.