SUMMARYThe proton-translocating inorganic pyrophosphatases (H⁺-PPases) are primary electrogenic H⁺pumps that derive energy from the hydrolysis of inorganic pyrophosphate (PPi). They are widely distributed among most land plants and have also been found in several species of protozoan parasites. Here we describe, for the first time, the molecular cloning and functional characterization of a gene encoding an H⁺-pyrophosphatase in the protozoan scuticociliate parasitePhilasterides dicentrarchi, which infects turbot. The predictedP. dicentrarchiPPase (PdPPase) consists of 587 amino acids of molecular mass 61·7 kDa and an isoelectric point of 5·0. Several motifs characteristic of plant vacuolar H⁺-PPases (V–H⁺-PPases) were also found in the PdPPase, which contains all the sequence motifs of the prototypical type I V–H⁺-PPase fromArabidopsis thalianavacuolar pyrophosphatase type I (AVP1) plant. The PdPPase has a characteristic residue that determines strict K⁺-dependence, but unlike AVP1, PdPPase contains an N-terminal signal peptide (SP) sequence. Antibodies generated by vaccination of mice with a genetic or recombinant protein containing a partial sequence of the PdPPase and a common motif with the polyclonal antibody PAB_HKspecific to AVP1 recognized a single band of about 62 kDa in western blots. These antibodies specifically stained both vacuole and the alveolar membranes of trophozoites ofP. dicentrarchi. H⁺transport was partially inhibited by the bisphosphonate pamidronate (PAM) and completely inhibited by NaF. The bisphosphonate PAM inhibited both H⁺-translocation and gene expression. PdPPase and PAM also inhibitedin vitrogrowth of the ciliates. The apparent lack of V–H⁺-PPases in vertebrates and the parasite sensitivity to PPI analogues may provide a molecular target for developing new drugs to control scuticociliatosis.