It has been suggested that electrostatic interactions are critical for binding of retinoic acid by cellular retinoic acid-binding proteins (CRABP-I and CRABP-II). However, the roles of two conserved arginine residues (Arg-111 and Arg-131 in CRABP-I; Arg-111 and Arg-132 in CRABP-II) that interact with the carboxyl group of retinoic acid have not been evaluated. A novel competitive binding assay has been developed for measuring the relative dissociation constants of the site-directed mutants of CRABPs. Arg-111 and Arg-132 of CRABP-II were replaced with methionine by site-directed mutagenesis. The relative dissociation constants of R111M and R132M (Kd (R111M)/Kd (CRABP-II) and Kd (R132M)/Kd(CRABP-II)) were determined to be 40-45 and 6-8, respectively. The ring protons of the aromatic residues of the wild-type CRABP-II and the two mutants were sequentially assigned by two-dimensional homonuclear NMR in conjunction with three-dimensional heteronuclear NMR. Detailed analysis of the nuclear Overhauser effect spectroscopy spectra of the proteins indicated that the conformations of the two mutants are highly similar to that of the wild-type CRABP-II. These results taken together showed that Arg-111 and Arg-132 are important for binding retinoic acid but contribute to the binding energy only by approximately 2.2 and 1.2 kcal/mol, respectively. In addition, the relative dissociation constant of CRABP-II and CRABP-I (Kd (CRABP-II)/Kd (CRABP-I)) was determined to be 2-3, in close agreement with that calculated using the apparent Kd values determined under the same conditions by fluorometric titrations.