The binding of the lipophilic nutrients, retinal, vitamin D 2 , and retinyl palmitate by β-lactoglobulin was measured by analysis of changes in the fluorescence of the tryptophanyl residue of β-lactoglobulin or the retinyl moiety. The fluorescence intensity of the tryptophanyl residue was quenched by retinoid or vitamin D binding but was enhanced by palmitate binding. The analysis of competitive binding experiments with palmitate indicated that retinal and palmitate did not compete for the same site; however, vitamin D 2 , which binds with a stoichiometry of 2, appeared to displace palmitate at higher concentrations. Also, the retinoids and vitamin D 2 were bound more tightly than was palmitate. The results are consistent with the model in which the retinoids and vitamin D 2 bind in the calyx formed by the β-barrel; palmitate and a second molecule of vitamin D 2 bind in a surface pocket near the dimer contact region. Retinyl palmitate, which has both moieties, appeared to bind at both sites.