The polygalacturonate lyase and pectinesterase activities of Clostridium multifermentans, both produced extracellularly when the organism grows on pectin or polygalacturonate, have been suggested to be associated in a single complex. Both enzymic sites act on their respective substrates by single-chain action patterns, as shown by equivalent release of terminal tritium label and total product throughout the reaction. From these results, the Km and V of the lyase, and the amount of lyase activity present, we calculate the steady-state concentration of lyase substrate expected during action of the two sites on pectin if the sites are independent. No such steady-state concentration of lyase substrate was observed. Therefore, we conclude that the two types of active site act in a coordinated manner; the polysaccharide chain passes from the esterase site to the lyase site without intermediate dissociation and rebinding. This 'molecular disassembly line' constituted by the two sites may represent a system of general significance in synthesis and degradation of biological polymers.