The fluorescent probe 1-anilinonaphthalene 8-sulfonic acid (1,8-ANS) has been used to characterize a general assay for members of the intracellular lipid-binding protein (iLBP) multigene family. The adipocyte lipid-binding protein (ALBP), the keratinocyte lipid-binding protein (KLBP), the cellular retinol-binding protein (CRBP), and the cellular retinoic acid-binding protein I (CRABPI) have been characterized as to their ligand binding activities using 1,8-ANS. ALBP and KLBP exhibited the highest affinity probe binding with apparent dissociation constants (K(d)) of 410 and 530 nM, respectively, while CRBP and CRABPI bound 1,8-ANS with apparent dissociation constants of 7.7 and 25 μM, respectively. In order to quantitate the fatty acid and retinoid binding specificity and affinity of ALBP, KLBP, and CRBP, a competition assay was developed to monitor the ability of various lipid molecules to displace bound 1,8-ANS from the binding cavity. Oleic acid and arachidonic acid displaced bound 1,8-ANS from ALBP, both with apparent inhibitor constants (K(i)) of 134 nM, while all-trans-retinoic acid exhibited a sevenfold lower K(i) (870 mM). The short chain fatty acid octanoic acid and all-trans-retinol did not displace the fluorophore from ALBP to any measurable extent. In comparison, the displacement assay revealed that KLBP bound oleic acid and arachidonic acid with high affinity (K(i) = 420 and 400 nM, respectively) but bound all-trans-retinoic acid with a markedly reduced affinity (K(i) = 3.6 μM). Like that for ALBP, neither octanoic acid nor all- trans-retinol were bound by KLBP. Displacement of 1,8-ANS from CRBP by all- trans-retinal and all-trans-retinoic acid yielded K(i) values of 1.7 and 5.3 μM, respectively. These results indicate the utility of the assay for characterizing the ligand binding characteristics of members of the iLBP family and suggests that this technique may be used to characterize the ligand binding properties of other hydrophobic ligand binding proteins.