The catalytic subunit of a membrane-bound pyrophosphatase was purified by electroendosmotic preparative electrophoresis from etiolated pea stem mitochondria. The enzyme was identified as a single peak relatively pure, because only a very limited number of polypeptides were detectable by SDS/PAGE of the active fractions. The pyrophosphatase was associated to a band with a molecular mass of 35 kDa, showing a specific activity of 0.7 mumol Pi . mg-1 protein . min-1 (37 degrees C, pH 8.0) and an apparent Km value of 200 microM. The hydrolytic activity required Mg2+, was inhibited by imidodiphosphate (HNO6P2Na4), Ca2+, F- and was stimulated by phospholipids. Cardiolipin, phophatidylcholine and phosphatidylethanolamine had the maximal activating effect. The isolated protein is very similar to the catalytic subunit of pyrophosphatases isolated from rat liver (beta-subunit) and Saccharomyces cerevisiae mitochondria.