Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) specifically cleaves GPIs. This phospholipase D is a secreted protein consisting of two domains: an N-terminal catalytic domain and a predicted C-terminal β-propeller. Although the biochemical properties of GPI-PLD have been extensively studied, its catalytic site has not been identified. We hypothesized that a histidine residue(s) may play a critical role in the catalytic activity of GPI-PLD, based on the observations that (i) Zn 2+ , which utilizes histidine residues for binding, is required for GPI-PLD catalytic activity, (ii) a phosphohistidine intermediate is involved in phospholipase D hydrolysis of phosphatidylcholine, (iii) computer modelling suggests a catalytic site containing histidine residues, and (iv) our observation that diethyl pyrocarbonate, which modifies histidine residues, inhibits GPI-PLD catalytic activity. Individual mutation of the ten histidine residues to asparagine in the catalytic domain of murine GPI-PLD resulted in three general phenotypes: not secreted or retained (His 56 or His 88 ), secreted with catalytic activity (His 34 , His 81 , His 98 or His 219 ) and secreted without catalytic activity (His 29 , His 125 , His 133 or His 158 ). Changing His 133 but not His 29 , His 125 or His 158 to Cys resulted in a mutant that retained catalytic activity, suggesting that at least His 133 is involved in Zn 2+ binding. His 133 and His 158 also retained the biochemical properties of wild-type GPI-PLD including trypsin cleavage pattern and phosphorylation by protein kinase A. Hence, His 29 , His 125 , His 133 and His 158 are required for GPI-PLD catalytic activity.