Glucose phosphate isomerase (GPI) was purified from human placenta utilizing cross-linked spherical particle phosphocellulose. In three steps, GPI could be purified approximately 5500 fold with greater than 50% recovery. The purified enzyme exhibited four bands upon non-denaturing PAGE and isoelectric focusing (IEF) when stained with GPI specific activity stain. The four isozymes were isolated by preparative IEF. The isoelectric points of the isozymes were determined. Sodium dodecyl sulfate (SDS) gel electrophoresis showed two types of subunits with different molecular weights. Structural analyses showed both types of subunits had blocked amino termini. Other properties of the isozymes and subunits, including immunological reactivity, pH stability, peptide mapping and amino acid composition, were also established.