BACKGROUND Serine and glycine are major one-carbon donors. In eukaryotic cells one-carbon metabolism is compartmentalized; the glycine cleavage enzymes are mitochondrial, while the cytosolic and mitochondrial forms of serine hydroxymethyltransferase (SHMT) are independently transcribed. DESIGN One-carbon donors ([3-13C]serine, [2, 3, 3-d3]serine, or [2-13C]glycine) were administered in separate infusions to healthy males (n = 5). Monocyte DNA was subsequently isolated for the determination of deoxynucleotide enrichment by LC-MS/MS. RESULTS Total thymidine and deoxyadenosine enrichment was similar for [3-13C]serine and [2, 3, 3-d3]serine [Table]. M+2 thymidine enrichment (i.e., that deriving from cytosolic SHMT) constituted 23% of [2, 3, 3-d3]serine derived enrichment. Thymidine enrichment from [2-13C]glycine was lower than from either labeled serine while deoxyadenosine enrichment from [2-13C]glycine was overestimated, as [2-13C]glycine was also directly incorporated into purines. Percentage molar enrichment (mean ± SD) of thymidine (dT) and deoxyadenosine (dA). Values in brackets are molar enrichments expressed as a percentage of total one-carbon enrichment: {enrichment / (enrichment from [2, 3, 3-d3]serine + enrichment from [2-13C]glycine)} CONCLUSION The majority (~84%) of one-carbon units used for thymidine synthesis are mitochondrial derived. Supported by NIH DK56274 and GCRC grant M01-RR00082