AbstractPaper chromatography of pectin‐methylesterase and polygalacturonase produced byClostridium felsineumwas achieved with buffers, buffered salt solutions and acetone‐water mixture. No true separation of both enzymes was attained and they became inactivated when chromatographed on paper under appropriate conditions. The chromatographed enzyme preparation showed only pectin‐methylesterase activity when the acetone‐water solvent was used as the developing agent. The aforementioned enzyme occupied the lower part of the chromatograms on using the ascending technique. The inactivation of pectolytic enzymes was attributed to their denaturation by spreading in the form of a film on the paper chromatograms. The extent of solubility of these enzymes in the developing agent was found to be a limiting factor for their spreading on the chromatograms and, hence, their inact