This report describes the purification and partial characterization of a novel retinoic acid-binding protein (CRABP-II) from neonatal rat pups. The isolation procedure included gel filtration on Sephadex G-75, ion exchange chromatography on DEAE-cellulose, and high performance liquid chromatography (HPLC) on a DEAE 5PW column. Two retinoic acid-binding peaks were resolved at the DEAE-cellulose step, with CRABP-I in the major peak and CRABP-II in the minor peak. Apparent homogeneity was achieved for both binding proteins after the HPLC step. CRABP-II consists of a single polypeptide, migrating with an apparent Mr of 15,000 in sodium dodecyl sulfate-polyacrylamide gel. It has an isoelectric point of 5.0. The dissociation constant for CRABP-II of retinoic acid was estimated to be 65 nM by fluorescence titration. Amino-terminal sequence analysis showed that CRABP-II has a distinct sequence, while the CRABP-I sequence is exactly identical to that of the rat testis CRABP. Despite the extensive sequence homology between CRABP-I and CRABP-II, antibodies directed against CRABP-I did not cross-react with CRABP-II.