The folate binding proteins (FBPs) of KB cells which were cultured in normal (N) and folate-deficient (D) medium have been characterized. The 200,000 g supernate of lysed cells contained two FBPs which could be separated by DEAE-Bio-Gel A chromatography, indicating that they differ in ionic charge although they could not be separated by gel filtration through Sephadex G-100 (apparent Mr approximately 40,000). Two species of FBP, a major form of apparent Mr approximately 160,000 and a minor form of apparent Mr approximately 40,000, were identified by gel filtration through Sephadex G-150 in the membrane component of the cells after solubilization with Triton X-100. An additional FBP was isolated and purified by affinity chromatography from the medium in which these cells were cultured. By gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent Mr of this FBP was approximately 44,000. The association constants for pteroylglutamic acid of the FBPs in the 200,000 g cell lysate supernate, culture medium, and Triton-solubilized membrane were similar and the relative affinity of folate analogs for the FBP, vis-à-vis pteroylglutamic acid, was similar for all species. An antiserum raised to the purified FBP from the culture medium precipitated the FBPs in the 200,000 g cell lysate supernate, Triton-solubilized membrane, and culture medium, indicating antigenic homology among these FBPs. There was no unsaturated FBP in the 200,000 g cell lysate supernate or medium when KB cells were cultured in N medium. However, when cells were cultured in D medium, the unsaturated FBP of the 200,000 g cell supernate and culture medium was substantial (9.2 and 14.1 pmol/mg protein, respectively). Unsaturated FBP was detected in the membrane of normal cells but this also increased when these cells were cultured in D medium (4.5 to 756 pmol/mg protein), indicating that the FBPs of these cellular compartments are normally saturated by folate. After 16 weeks of culture in D medium, the total folate binding capacity of the membrane-associated FBP was twofold greater than that of normal KB cells, indicating the induction of FBP.