The nature and density of charged sites in an agarose gel have been studied. Diffusion and partition coefficients of various organic and inorganic ions have been measured as a function of ionic strength, mu, and pH to investigate the solute-gel interactions resulting from charge effects and specific complexation. The majority of binding sites in the gel are pyruvate groups, with an intrinsic protonation constant of log K-a(int) = 3.9. We measured the charge density of the gel as a function of added salt and pH and evaluated the Donnan potential. The partition coefficients of cations decrease and those of anions increase with increasing ionic strength because of progressive screening of the anionic sites in the gel, as predicted by the Boltzmann and Poisson-Boltzmann equations. The charges in the get become completely screened at mu approximate to 10(-2). As predicted with the Smoluchowski-Poisson-Boltzmann theory, the diffusion coefficient of cationic species is reduced at low ionic strength. We tested both cylindrical and spherical symmetries of Poisson-Boltzmann cell models to describe these variations and obtained better results with the former. In addition to electrostatic effects, we detected specific interactions between metal ions and the gel, and we determined the intrinsic association constants. General models are presented for the partitioning and diffusion of solutes in the gel, which consider steric, electrostatic, and chemical interactions.