The molecular weight of delta-5-3-ketosteroid isomerase from Pseudomonas testosteroni was determined by means of sedimentation equilibrium and exclusion chromatography over a wide range of enzyme concentrations in 0.2 M potassium phosphate buffer, pH 7.0. In addition, the sedimentation constant of the enzyme was determinded over an extended range of concentrations. The enzyme was found to have a molecular weight of 26,000 plus or equal to 1,000, suggesting that it is a dimer of identical or similar 13,400 molecular weight polypeptide chains. In the ultracentrifuge this dimeric species was found to undergo aggregation at enzyme concentrations above 2 mg per ml and dissociation at enzyme concentrations below 0.05 mg per ml. Exclusion chromatography studies indicate that under the conditions of chromatography the oligomeric enzyme is partially dissociated at enzyme concentrations in the range 0.2 to 0.002 mug per ml. These results suggest that under conditions of enzyme assay in 0.2 M potassium phosphate buffer, pH 7.0, isomerase is in a monomeric state of aggregation.