Cell-free extracts of a strain of Proteus vulgaris degrade NADH to reduced nicotinamide riboside, adenosine and two molecules of phosphate. The system is weakly active in fresh cell extracts, but activity is increased about 10-fold on rapid heating to 70-100 degrees C. On returning to room temperature, the activity returns rapidly to its initial low value but can be re-activated by again heating to 70-100 degrees C. Reversible activation can also be effected by extremes of pH or by teatment with 8M-urea. Activation appears to be due to reversible changes in conformation of the protein of the enzyme rather than to combination of the enzyme with a heat-labile inhibitor. The active form can be stabilized by addition of PPi. The system, which also possesses 5'-nucleotidase activity not separable from the NADH pyrophosphatase, requires Co2+ (0.4mM) for maximum activity. Although activated at relatively high temperatures, it is not enzymically active until cooled to 50-60 degrees C. It may be purified by affinity chromatography (with NAD+ as ligand) to an activity over 400 times that of the crude cell extract, and yields only one major band on polyacrylamide-gel electrophoresis.