Cellular retinol-binding protein I (CRBP I) and cellular retinol-binding protein II (CRBP II) are closely homologous proteins that play distinct roles in the maintenance of vitamin A homeostasis. The solution structure and dynamics of CRBP I and CRBP II were compared by multidimensional NMR techniques. These studies indicated that differences in the mean backbone structures of CRBP I and CRBP II were localized primarily to the alphaII helix. Intraligand NOE cross-peaks were detected for the hydroxyl proton in the NOESY spectrum of CRBP I-bound retinol, but not for CRBP II-bound retinol, indicating that the conformational dynamics of retinol binding are different for these two proteins. As determined by Lipari-Szabo formalism, both the apo and holo forms of CRBP I and CRBP II are conformationally rigid on the pico- to nanosecond timescale. transverse relaxation optimized spectroscopy-Carr-Purcell-Meiboom-Gill -based 15N relaxation dispersion experiments at both 500 MHz and 600 MHz magnetic fields revealed that 84 and 62 residues for apo-CRBP I and II, respectively, showed detectable conformational exchange on a micro- to millisecond timescale, in contrast to three and seven residues for holo-CRBP I and II, respectively. Thus binding of retinol markedly reduced conformational flexibility in both CRBP I and CRBP II on the micro- to millisecond timescale. The 15N relaxation dispersion curves of apo-CRBP I and II were fit to a two-state conformational exchange model by a global iterative fitting process and by an individual (residue) fitting process. In the process of carrying out the global fit, more than half of the residue sites were eliminated. The individual chemical exchange rates k(ex), and chemical shift differences, Deltadelta, were increased in the putative portal region (alphaII helix and betaC-betaD turn) of apo-CRBP II compared to apo-CRBP I. These differences in conformational flexibility likely contribute to differences in how CRBP I and CRBP II interact with ligands, membranes and retinoid metabolizing enzymes.