The concentration of folate in erythrocytes was determined using a two-phase ligand-binding radioassay procedure described previously for measuring serum folate. The mean (± SD) folate concentration in erythrocytes of 20 normal subjects was 210 ± 57 ng/ml. In 12 patients clinically folate deficient who had normal serum B12 concentration, the mean (± SD) erythrocyte folate was 71 ± 39 ng/ml. Incubation of the lysed erythrocytes for 2 hr prior to boiling increased the radioassayable folate. The radioassayable folate decreased rapidly if the whole blood was stored at 4°C without ascorbate. Extracts of blood prepared with ascorbate could be stored at -20°C for several days. The radioassayable concentration of erythrocyte folate was similar to the values obtained using Lactobacillus casei when the concentration was 200 ng/ ml or less. With values higher by L. casei, the radioassayable folate was significantly lower even though the normal and folate-deficient groups were distinctly separated. This radioassay provides a rapid and reliable method of measuring erythrocyte folate, a parameter which reflects folate stores more reliably than serum folate concentration.