A putative cellular function of tetrahydropteridines (l-erythro-tetrahydrobiopterin and d-threo-tetrahydrobiopterin) was investigated in Dictyostelium discoideum Ax2 using a mutant disrupted in the gene encoding sepiapterin reductase (SR). The SR mutant, which produces about 3% of tetrahydropteridines if compared to wild-type, was elucidated to have several functional defects related to mitochondria and oxidative stress: retarded growth, poor spore viability, impaired mitochondrial function, and increased susceptibility to oxidative stress induced by hydroxylamine or cumene-hydroperoxide. However, the physiological defects were almost completely rescued by extrachromosomal expression of Dictyostelium SR. The results strongly suggested that tetrahydropteridines in Dictyostelium are associated with mitochondrial function, probably via direct protection against oxidative stress.