OBJECTIVE: To evaluate the impact of Peru's national folic acid fortification program on folic acid content in wheat flour, and the effect on birth prevalence of neural tube defects (NTDs) in Lima, and to compare the program's legislative requirements with international standards. METHODS: Bread was sampled from six sites across Peru and tested for folic acid. Data were obtained from the largest obstetric hospital in Lima on the prevalence of births (live and still) with NTDs during both the pre-fortification period (2004-2005) and post-fortification years (2007-2008). RESULTS: Folic acid content in the sampled bread met national legislative requirements but was less than one-half of the level recommended for Peru by the World Health Organization (WHO) (2.6 mg/kg wheat flour). Birth prevalence of NTDs was 18.4/10 000 in the pre-fortification period and 20.0/10 000 during post-fortification years. Relative risk for NTDs after fortification was 1.02 (95% confidence interval 0.77-1.35, P = 0.90). CONCLUSIONS: Peruvian legislative requirements for folic acid fortification are below international (WHO) recommendations; birth prevalence of NTDs in Lima is higher than international benchmarks; and no decrease in NTDs following fortification of flour with folic acid (according to Peruvian national standards) was observed. As increasing the level of folic acid in flour remains the most sustainable way of preventing NTDs, it is recommended that Peru increase its folic acid fortification requirements to meet those recommended by WHO (2.6 mg/kg).