Characteristics of iron binding to the solubilized brush border membrane (BBM) of rat intestine were studied. Specific Fe(II) and Fe(III) binding sites were detected by iron binding assay using immobilized BBM from the upper intestine on a nitrocellulose sheet and the binding to Fe(II) was considerably higher than that to Fe(III). The dissociation constants for Fe(II) were 0.26+/-0.02 nm (M+/-SE) for high-affinity binding sites and 2.70+/-0.26 nm forlow-affinity binding sites. For Fe(II),the number of high-affinity binding sites was 0.35+/-0.04 x 10(16)/mg of protein (M+/-SE) and that of low-affinity binding sites was 1.07+/-0.11 x 10(16)/mg of protein. Bound Fe(II) could not be replaced with Fe (II), Fe(III) or transferrin-bound iron and approximately 50% of bound Fe(II) was removed by chelators. The apparent molecular weights of Fe(II) binding peaks were 250 and 120 kDa by gel filtration. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) detected three bands with iron-binding activity. The distribution of the Fe(II) binding sites showed that the number of low-affinity sites was significantly higher in the proximal third of the intestine compared with the rest of the intestine. Iron deficiency increased the number of Fe(II) binding sites. These findings suggest that the Fe(II) binding sites might play a physiological role in iron absorption in the rat.