Radioactive methylfolate (14C-5CH3H4PteGlu) (10–14 μg/Kg) was fed to four lactating women presenting with breast abscesses necessitating cessation of lactation. The appearance of radiofolate in milk, plasma and urine over the next 24 hours was investigated. In spite of a minimal postabsorption rise of plasma biofolate, plasma radiofolate (including a dialysis-resistant (bound) fraction) increased steadily to 1.26 to 5.11 μg/l at 24 hours. Urinary radiofolate excretion was considerable. Total milk biofolate rose significantly by 15 to 28 μ/l, in contrast with a much smaller radiofolate fraction (1.95–3.88 μg/l) which at 24 hours was comparable with that of plasma. Milk radiofolate included a dialysis-resistant fraction rising to 0.75 to 1.15 μg/l at 24 hours. On chromatography (Sephadex-DEAE-A50) plasma, urine and milk showed a nonbound radiofolate peak suggestive of 10-CHO H4PteGlu. This folate may originate predominantly from the apocrine mammary glands. The in vivo labelled radiofolate milk binder could not be identified chromatographically, but it was shown that the in vitro milk binders of PteGlu and SCH3H4PteGlu could be separated chromatographically.