The interactions with retinol and retinol analogs of bovine cellular retinol-binding protein (CRBP) have been investigated, by means of fluorescence titrations, to obtain more information on the structural features of retinoid that may be required for their interaction with the binding protein. An approximately stoichiometric binding of retinol to bovine CRBP (K'd approximately 2 nM) has been found in direct binding assays. Although retinal exhibited relatively high binding affinity to bovine CRBP (K'd approximately 30 nM), a large excess of the retinoid could not compete with retinol for the carrier protein. On the assumption that retinol and retinal interact with the same binding site, this result indicates that the above-mentioned apparent dissociation constant for retinol.CRBP may be an overestimate and that its value may be as low as 0.1 nM. The finding of an exceedingly tight binding of retinol to CRBP provides further support for the possible role of CRBP-bound retinol, rather than its uncomplexed labile form, as substrate of enzymes involved in the metabolism of the vitamin. The results of these and previous studies indicate that CRBP is particularly sensitive to modifications of the retinol hydroxyl end group. Axerophthene, a retinol analog bearing a hydrogen atom in place of the hydroxyl end group, and beta-ionone exhibit rather low binding affinities for CRBP (K'd approximately 0.2 microM and approximately 4 microM, respectively), suggesting that the hydroxyl group and isoprene tail moieties contribute substantially to the retinol-binding affinity and specificity. These findings are consistent with the indications emerging from the three-dimensional structure determination of retinol.CRBP [Cowan, S. W., Newcomer, M. E. & Jones, T. A. (1993) J. Mol. Biol. 230, 1225-1246]. Additionally, the bulky end groups of fenretinide and N-ethyl retinamide replacing the retinol hydroxyl group have been found to prevent retinoid binding to CRBP. The primary structure of bovine CRBP has been determined and is highly similar to the structures of both human and rat CRBP (97% and 95% identical, respectively).