The observation that decreased thymidylate supply in vitro induces the expression of the Xq27 chromosome fragile site prompted us to examine cellular thymidylate metabolism. Using a sensitive enzyme assay for deoxyribonucleotide triphosphates, we found that the total cellular thymidine triphosphate pools in cell lines from fragile X patients and carriers do not differ from normal controls under either basal or folate-deficient conditions. This agrees with our earlier observation that the thymidylate synthase enzyme activities in crude cell extracts of five fragile X syndrome lymphoblast lines do not differ from those in normal controls under standard assay conditions. Although a difference in the amount of thymidine triphosphate available at the replication fork for DNA synthesis remains a possibility, our results indicate that a readily demonstrable defect in thymidylate metabolism is not present in fragile X syndrome cells.