In biological fluids IGF-I and IGF-II are bound to specific, high-affinity binding protein (BPs). Two human BPs have been isolated, one from serum, which is GH-dependent, the other from amniotic fluid (AF BP), and their cDNAs have recently been cloned. We report here the isolation of another, new species from cerebrospinal fluid (CSF) where this BP predominates. The protein was purified to homogeneity by a four-step procedure: gel filtration, chromatofocusing, hydrophobic-interaction chromatography and reverse-phase chromatography. Thereafter, SDS-polyacrylamide gel electrophoresis gave an Mr of 34,000 (non-reduced), chromatofocusing gave an isoelectric point of 5.0m and its affinity for IGF-II (3 x 10(10) M-1) was 10 times that for IGF-I. The N-terminal amino acid sequence of the first 15 residues determined in a BP preparation from the CSF of children was Leu-Ala-Pro-Gly-(/)-Gly-Gln-Gly-Val-Gln-Ala-Gly-Ala-Pro-Gly. A similar sequence was found for adult CSF, apart from residues 12 and 13 (-Leu-Leu-). These are highly analogous with the sequences starting from residue 69 of the GH-dependent BP, and from residue 61 of the AF BP. The new BP isolated is therefore related to, but distinct from, the other human BPs.