Electron paramagnetic resonance (EPR) is here used to study the interaction between gum arabic and a fatty acid. The EPR spectra of 5-doxyl stearic acid (5-DSA), a spin-labeled fatty acid analog, displayed increasingly anisotropic line features upon addition of gum arable, indicating a strong immobilization of the nitroxyl moiety when the fatty acid is bound to gum arabic. To understand the nature of the interaction, EPR measurements were carried out at different pHs and using two fractions of gum arabic separated by hydrophobic interaction chromatography (HIC). 5-DSA bound favorably to the hydrophobic fraction, which contains mainly glycoprotein, and a small amount of high molecular weight arabinogalactan protein (AGP). Binding occurred to a less extent to the hydrophilic fraction, which contains essentially arabinogalactan (AG). Such a hydrophobic binding mechanism is further supported by a sharp drop in the binding when pH is raised above the pK(a) value of 5-DSA (similar to pH 5). This is because the ionization of carboxylic groups would lead to increased polarity and hydrophilicity of the fatty acid. A secondary effect involving the formation of ionic hydrogen bonds between carboxylic groups in fatty acid and lysine residues in gum arabic might also contribute. This is consistent with the reduction in binding ability when the pH was elevated above the pK(a) value of lysine residue (similar to pH 10). The biological significance of these findings is considered.