To evaluate folate-dependent carbon incorporation into the purine ring, we measured 13C-enrichment independently at C2 and C8 of urinary uric acid (the final catabolite of purines) in a healthy male after an independent oral dose of [6RS]-5-[13C]-formyltetrahydrofolate ([6RS]-5-H13CO-H4folate) or 10-H13CO-7,8-dihydrofolate (10-H13CO-H2folate). The C2 position was 13C-enriched more than C8 after [6RS]-5-H13CO-H4folate, and C2 was exclusively enriched after 10-H13CO-H2folate. The enrichment of C2 was greater from [6RS]-5-H13CO-H4folate than 10-H13CO-H2folate using equimolar bioactive doses. Our data suggest that formyl C of [6RS]-10-H13CO-H4folate was not equally utilized by glycinamide ribotide transformylase (enriches C8) and aminoimidazolecarboxamide ribotide (AICAR) transformylase (enriches C2), and the formyl C of 10-H13CO-H2folate was exclusively used by AICAR transformylase. 10-HCO-H2folate may function in vivo as the predominant substrate for AICAR transformylase in humans. © 2007 Elsevier Inc. All rights reserved.