The tendency of equine IgG to form soluble aggregates at low pH was a function of elution time during purification by anion exchange chromatography on DEAE-Sepharose, with later eluting fractions showing faster aggregation rate and higher aggregate levels in the pH range 3.5-4.0. An ELISA assay using antibodies to different equine IgG subclasses was used to show that subclasses have different elution profiles, with IgGb being slowest to elute. Separation of the soluble aggregates by polyethylene glycol precipitation and analysis for subclass composition showed that aggregates were enriched in IgGb. ANS binding experiments demonstrated that later eluting fractions have a considerably greater exposure of surface hydrophobic groups at pH 3.7. The results underscore the importance of careful control of the ’cut-off’ volume during ion exchange chromatography of polyclonal IgG in preparing a consistent purified product.