The chromatographic properties of Superdex 30 prep grade medium have been investigated in non-denaturing and denaturing mobile phases using commercially available proteins and peptides as well as low-molecular-mass (M(r)) recombinant polypeptides. The medium is a macroreticular gel composed of crosslinked agarose beads to which dextran has been covalently bound. The mean particle size is approximately 34 microns. Experimental results show a linear relation between the distribution coefficient (KD) and the log10 M(r) in the fractionation range 24,000-3000. The relationships between resolution and flow-rate or load volume were investigated and shown to be comparable with those of Superdex 75 and 200 prep grade media. Minimal loss of resolution occurred in the flow-range from 30-60 cm/h. Load volumes of up to 5% total column volume could be applied while maintaining baseline resolution of polypeptide mixtures. Non-specific interactions between the matrix and certain samples were characterized. The predominant interactions with the resin appear to be hydrophobic in nature rather than ionic. Hydrogen bonding may also play a role in the retardation of certain small molecules. The applicability of the resin for separating dimeric and oligomeric forms of low-molecular-mass recombinant proteins was shown.