Tetrahydrobiopterin (BH4) is a cofactor of a number of regulatory enzymes. Although there are no known BH4 exogenous sources, the tissue content of this biopterin increases with age in GTP cyclohydrolase 1-deficient hyperphenylalaninemia-1 (hph-1) mice. Since certain bacteria are known to generate BH4, we hypothesize that generation of this biopterin by the intestinal microbiota contributes to its tissue increase in hph-1 adult mice. The goal of this study was to comparatively evaluate hph-1 mice and wild-type C57Bl/6 controls for the presence of intestinal BH4-producing bacteria. Newborn and adult mice fecal material was screened for 6-pyruvoyltetrahydropterin synthase (PTPS-2) an enzyme only present in BH4-generating bacteria. Adult, but not newborn, wild-type control and hph-1 mouse fecal material contained PTPS-2 mRNA indicative of the presence of BH4-generating bacteria. Utilizing chemostat-cultured human fecal bacteria, we identified the PTPS-2-producing bacteria as belonging to the Actinobacteria phylum. We further confirmed that at least two PTPS-2-producing species, Aldercreutzia equolifaciens and Microbacterium schleiferi, generate BH4 and are present in hph-1 fecal material. In conclusion, intestinal Actinobacteria generate BH4. This finding has important translational significance, since manipulation of the intestinal flora in individuals with congenital biopterin deficiency may allow for an increase in total body BH4 content.