White Leghorn hens were fed purified folate-deficient diets or commercial corn- and soybean meal-based diets supplemented with different amounts of folic acid. The folate contents of egg yolk and blood plasma from these hens were estimated with an isotope-dilution, radioligand-binding assay. Folates in egg yolk were concentrated approximately 43-fold relative to the blood plasma from which they were derived. Yolk and plasma folate concentrations became saturated with increasing dietary folate. Hens fed a commercial, folate-sufficient diet (0.72 mg folate/kg) produced eggs with slightly less than half of the maximal folate content. Based on tritium deposition in egg yolk and egg white, the biological half-life of [3H]folic acid injected intraperitoneally into two folate-sufficient hens was approximately 15 days, while it was > or = 40 days in two hens fed a purified folate-deficient diet (0.07 mg folate/kg) that also reduced egg production. Radioactivity in egg yolk was concentrated more than 100-fold relative to egg white in both cases. The [3H]folates remaining in the hens at the end of the experiment were substantially more concentrated in liver than in kidney, heart, or skeletal muscle. The specific radioactivity of folates in the liver of folate-deficient hens after 78 days was almost 10 times greater than in folate-sufficient hens after 39 days. Laying hens have highly efficient conservation and delivery systems for folates.