A membrane-associated iron-binding complex was isolated from rat intestinal mucosa during iron absorption. Two 59Fe peaks (peaks 1 and 2) were separated on Sepharose 6B gel filtration from detergent solubilized 20,000 x g precipitate of upper intestinal mucosal homogenate after administration of 59Fe-labelled ferrous materials. Peak 1 was a membrane iron-binding complex whose apparent molecular weight was over 10(6) Da estimated by gel filtration, while peak 2 was identified as ferritin. Three major bands were detected on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of peak 1. The treatment of mucosal homogenate with 5 mM ethylenediaminetetraacetic acid released 59Fe binding from peak 1. Incorporation of 59Fe to peak 1 showed the maximum at 1 h and then reduced, while [59Fe]ferritin showed reciprocal behavior, which suggested that peak 1 may be a rapid turnover iron pool and transfer 59Fe to ferritin. Peak 1 was also isolated from the brush border membrane and showed similar SDS-PAGE pattern to that from the 20,000 x g precipitate of mucosal homogenate. Western blot analysis did not reveal immunoreactive transferrin in peak 1. Those findings suggest that peak 1 may be a non-ferritin, non-transferrin iron-binding complex located on the brush border membrane and accept iron from the intestinal canal during iron absorption.