This paper describes the development of a new, malachite green based, enzymatic assay for the identification of specific inhibitors of inorganic pyrophosphatase (iPPase) from Saccharomyces cerevisiae for antifungal drug discovery. The human iPPase was used as counterscreen. The coding regions of both enzymes were amplified, cloned into a vector providing a His-tag at the C-terminus, expressed in Escherichia coli, and purified by metal chelate affinity chromatography. Since the complete human sequence had not been published previously, the human iPPase was cloned on the basis of expressed sequence tag data. The human sequence was confirmed and showed about 55% amino acid identity with the yeast enzyme and 95% identity with an already published bovine enzyme. Both recombinant iPPases were characterized with regard to their biochemical properties, showing that the His-tag did not influence the specific activity, pH optimum, inhibitor profile, or dimerization. The enzyme activity was determined by quantifying released phosphate by complex formation with malachite green. The resulting complex was quantified spectrophotometrically. The assay was adapted to a microtiter plate format, Thus, it is possible to screen a large compound pool for iPPase inhibitors in a short period of time.