Multiple forms of polygalacturonases are produced by Aspergillus carbonarius, Three forms of this enzyme differing in molecular weights (PG 1, 61,000; PG II, 42,000; and PG III, 47,000) and enzymatic properties have been isolated to apparent homogeneity by a simple method based on molecular sieve chromatography on Sephacryl S-200, ion exchange chromatography on CM Sephadex, followed by gel filtration on Sephadex G-50 superfine. All the three enzymes are endo-enzymes with high affinity for polygalacturonic acid. One of the forms, PG II, which accounts for >60% of the total polygalacturonases activity, had the highest reported specific activity of 7,000 U mg(-1) protein. The three enzymes differ in their amino acid composition, affinity toward substrate, sensitivity toward metal ions, and thermal stability. The midpoint of thermal inactivation temperature (T-m) was 43, 46, and 54 degrees C for PG I, PG II, and PG III, respectively. The thermal inactivation of the multiple forms followed first-order kinetics with estimated half-lives of 6, 10, and 32 min, respectively.