This paper describes a spectrophotometric assay that can measure the inorganic pyrophosphate produced from various enzymatic reactions. This is a coupled assay in which the addition of inorganic pyrophosphatase initially cleaves the pyrophosphate into two molecules of phosphate. The phosphate is then detected by the conversion of 2-amino-6-mercapto-7-methylpurine ribonucleoside to 2-amino-6-mercapto-7-methylpurine by purine nucleoside phosphorylase [M.R. Webb (1992) Proc. Natl. Acad. Sci. USA 89, 4884-4887]. The reaction is monitored by measuring the increase in absorbance at 360 nm. The generation of two molecules of phosphate from each molecule of pyrophosphate increases the sensitivity of the assay, which has a linear range from about 1 to 75 nmol pyrophosphate in a 1-ml reaction volume. To demonstrate the general usefulness of this assay, we show that the inorganic pyrophosphate generated by reactions involving acetyl-CoA synthetase and luciferase can be readily detected and that continuous as well as end-point assays can be performed.