Fifty piglets from birth to 14 days of age were used to investigate iron binding substances of neonatal intestinal mucosa, and to evaluate the effects of these substances in neonatal iron absorption. 59Fe-labeled ferric citrate with a molecular weight of 1,500 was injected directly into the ligated duodenum. Approximately 65% of radioiron in the whole homogenate of scraped intestinal mucosa was precipitate by centrifugation for 30 minutes at 10,000 X g. Over 70% of the radioiron of the supernatant applied to Sephadex G-200 column was eluted and separated into three radioiron fractions. These iron binding substances were identified as ferritin, transferrin and a low molecular weight form by elution characteristics on chromatography and by immunological technique. Although ferritin radioactivity was the major fraction of peak 1 (73%), transferrin activity was only 54% of the whole radioiron of peak 2. The sodium dodecyl sulfate (SDS) extract of saline insoluble particles from the mucosa applied to Sepharose 4B column eluted as a single peak near the point corresponded with the ferritin peak. Although the ferritin peak contained a higher percentage of the 59Fe than the transferrin peak at birth, the percentage of ferritin decreased and percentage of transferrin increased with age. A SDS soluble iron binding substance was found in the insoluble particles of mucosa of newborn as well as nursing piglets. Since SDS, as well as saline soluble, iron binding proteins were detected in the newborn intestinal mucosa, neonatal cell membrane and cytoplasma may have an active iron transfer system. Thus, it seems likely that neonatal mucosal cell has two active iron transport mechanisms: endocytosis and transport across the plasma membrane.