© 2015 by the authors; licensee MDPI, Basel, Switzerland. We determined whether ring-2 carbon of histidine is folate-dependently transferred to carbons 8 (C8) and/or 2 (C2) in urinary uric acid in humans. Two adults collected each urine void for four days. Aliquots of urine for the first day were used for baseline values; then the subjects ingested 0.7 g (3.3 mmol) of L-[ring-2-13C]histidine and collected urine for three experimental days. Aliquots were analyzed for percentage 13C-content at C2 and C8 by a liquid-chromatography-mass spectrometry method. Percentage enrichment was determined by subtracting time-of-day paired baseline percentage 13C-content from experimental percentage 13C-content for each void. C2 was predominantly 13C-enriched in the majority of voids. The percentage enrichments at C2 for two subjects were 0.14 (±0.028 [SEM], n = 26) and 0.18 (±0.049, n = 21), whereas at C8, they were 0.008 (±0.006) and −0.005 (±0.008), respectively. The mean C2-enrichments were significantly greater than zero (p < 0.01), whereas those of C8 were not (p > 0.2). The enrichment had a diurnal rhythm peaking in the morning. Our results may be useful in the estimation of the timing for the administration of drugs that interfere with purine nucleotide biosynthesis in the treatment of cancer and autoimmune disease.