Folate is an essential vitamin involved in a number of biological processes. High affinity folate binding proteins (FBPs) exist both as glycosylphosphatidylinositol-linked, membrane associated folate binding proteins and as soluble FBPs in plasma and some secretory fluids such as milk, saliva and semen. The function and significance of FBPs are unresolved, however, it has been suggested that they may facilitate folate uptake, e.g. during suckling. The present study shows that megalin, a large, multiligand endocytic receptor and member of the low-density lipoprotein-receptor family, is able to bind and mediate cellular uptake of FBP. Surface plasmon resonance analysis shows binding of bovine and human milk FBP to immobilized megalin, but not to low density lipoprotein receptor related protein. Binding of (125)I-labeled folate binding protein (FBP) to sections of kidney proximal tubule, known to express high levels of megalin, is inhibitable by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin. Immortalized rat yolk sac cells, representing an established model for studying megalin-mediated uptake, reveal (125)I-labeled FBP uptake which is inhibited by receptor associated protein and by antimegalin antibodies. Microinjection of (125)I-labeled FBP into renal tubules in vivo shows proximal tubular uptake by endocytosis. Megalin is expressed in several absorptive epithelia, including intestine and kidney proximal tubule, and thus the present findings provide a mechanism for intestinal and renal endocytic uptake of soluble FBP.