Supplementing laying hen diets with folic acid at 4 mg/kg of diet for 3 wk led to the production of folate-enriched eggs with an approximately 3-fold increase in egg folate concentration relative to a regular commercial egg. However, because long-term consistency in folate deposition is important for successful folate enrichment of natural foods such as eggs, 2 studies were designed to determine the level of folic acid required to maximize egg folate content and consistency throughout the entire cycle of egg production. In study 1, Hy-Line W36 and W98 laying hens (n = 156/treatment) received a barley-based diet containing 0 and 4 mg/kg of folic acid, whereas in study 2, a total of 1,248 Hy-Line CV20 laying hens (n = 416/treatment) received a barley-based ration supplemented with folic acid at 0, 2, or 4 mg/kg of diet. Both studies lasted for eleven 28-d periods. Response criteria included production parameters, egg quality measurements, and measures of egg folate content. Results of both long-term production studies support the use of 4 mg of folic acid/kg of diet as the recommended level of folic acid supplementation in terms of maximizing egg folate concentration over time. This is supported by the higher egg folate content and more consistent egg folate deposition at 4 mg of folic acid/kg of diet as compared with 0 and 2 mg of folic acid/kg of diet. Hy-Line W98, a strain of hens selected for large egg mass, benefited more from increased dietary folic acid in terms of increased egg production than did Hy-Line W36 and CV20 hens, strains selected for better egg efficiency and livability. Sensitivity and responsiveness to increasing dietary folic acid concentration in terms of egg folate content were, however, similar for all strains. In conclusion, eggs of laying hens were consistently enriched with folate by dietary supplementation with 4 mg of folic acid/kg of diet throughout the production cycle.