Folate binding activity of high affinity was identified in the particulate fractions of rat kidney homogenates. This binding activity cofractionated with alkaline phosphatase and maltase, two brush border membranes markers. With an enriched preparation of brush border membranes, freed of endogenous folate by acid treatment, the binding of [3Hlfolate was found to be saturable (Kb = 4.2 X 10-11 M) and rapid. Binding was optimal at pH 6. 4-7.7 [3H]folic acid into the left renal artery of dogs, 6-24% of the 3H was still retained in the kidney and could be flushed out into the urine by a large dose of unlabeled folic acid. Rubin et al. (3) showed that methotrexate, which inhibits folic acid reabsorption (1, 2), is taken up by rabbit renal cortical slices by two processes: one which is rapid and energy independent and a second which is slower and requires energy.On the basis of these observations it has been suggested (3, 4) that this initial step of folate reabsorption in the kidney involves the binding of filtered folate to a specific protein of the proximal tubular cells. Dihydrofolate reductase has been proposed as the binding protein because this enzyme is present in relative abundance in this tissue. An alternate possibility is that this cellular factor is a high-affinity binding protein of the type that has been recently identified in milk (5), blood serum (6-9), and various other tissues including the soluble fraction of porcine kidney homogenates (10-13). Such proteins are characterized by rapid association and slow dissociation with folic acid (Kb = 10-1o-10-11 M), a preference for folate mono-and polyglutamates over those with one carbon substitution at the 5 position or methotrexate (7), and different effects of pH on binding of folic acid and N5-methyltetrahydrofolate (14, 15 Schmitz et al. (17). In this procedure, CaCl2 was added to the homogenate to a final concentration of 10 mM. The mixture was stirred for 10 min and centrifuged at 2000 X g for 10 min, producing a "cell debris" precipitate (fraction P1). Centrifugation of the supernatant at 20,000 X g for 15 min resulted in the separation of soluble proteins (fraction S) from a second pellet (fraction P2). By electron microscopy, P2 appeared to be a highly enriched preparation of membrane vesicles. All fractions were assayed for protein (18), their capacity to bind radioactive folate (see below), and markers activities for brush border membranes (alkaline phosphatase and maltase) (19,20), mitochondria (succinate cytochrome c reductase) (21), basolateral membranes (Na+,K+-ATPase) (22), microsomes (glucose-6-phosphatase), and lysosomes (acid phosphatase) (23)