Objective: To validate a 2-tier approach for newborn screening (NBS) of remethylation defects. Study design: The original NBS dried blood spots of 5 patients with a proven diagnosis of a remethylation disorder and 1 patient with biochemical evidence of such disorder were analyzed retrospectively to determine disease ranges for methionine (Met; 4.7-8.1 μmol/L; 1 percentile of healthy population, 11.1 μmol/L), the methionine/phenylalanine ratio (Met/Phe; 0.09-0.16; 1 percentile of healthy population, 0.22), and total homocysteine (tHcy; 42-157 μmol/L; 99 percentile of normal population, 14.7 μmol/L). These preliminary disease ranges showed a sufficient degree of segregation from healthy population data, allowing the selection of cutoff values. A simple algorithm was then developed to reflex cases to a second-tier testing for tHcy, which has been applied prospectively for 14 months. Results: A total of 86 333 NBS samples were tested between January 2007 and March 2008, and 233 of them (0.27%) met the criteria for second-tier testing of tHcy. All cases revealed concentrations of tHcy <15 μmol/L and were considered unaffected. No false-negative results have been reported with a state-wide system based on 2 combined metabolic clinics and laboratories that cover the entire Minnesota population and border areas of neighboring states. Conclusions: Pending more conclusive evidence from the prospective identification of additional true-positive cases, NBS for remethylation disorders appears to be feasible with existing methodologies, with only a marginal increase of the laboratory workload.