Glycolate oxidase (GO) consists of identical subunits and therefore should show one definite pI value, but the isolated GO exhibited a range of pIs. This study investigated the underlying cause of this phenomenon. GO was purified and showed a molecular weight of 40 kDa by SDS-PAGE. Elution behavior on DEAE-cellulose chromatography and cellulose acetate membrane electrophoresis indicated that the purified GO was highly basic (pI>10.0). Repeated IEF and cIEF analysis showed that the pI of the purified GO was in the range of 10.0-3.25, either in a smear form or as distinct bands. 2-DE analysis showed that the 40 kDa subunit of GO displayed variable pIs from 9.6 to 3.65. It was likely that the purified GO was actually a complex consisted of GO and an unknown protein. CE-SDS, SDS-cellulose acetate membrane electrophoresis and amino add compositions indicated that the unknown protein was a highly basic polymer (BP) consisting of basic and phenylalanine-rich oligopeptide (BOP). Many BOPs are located on the surface of the acidic GO via ionic and hydrophobic interactions and formed GO-BOP complex (GC), resulting in a highly basic GC although GO itself was acidic. This hypothesis was further supported by the facts that anti-GC serum failed to recognize GO, and GC showed a peak at 257 nm although GO has few phenylalanine residues. Irregular and incomplete disassociation between GO and BOP was observed in IEF and cIEF, resulting in various intermediates with different ratios of GO/BOP, which could be the reason for the range of pIs observed for GO.