Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in mammalian plasma, but little is known of its cellular and tissue distribution. In this study frozen sections of perfused tissues from adult rats were stained with monoclonal antibodies against GPI-PLD. The most intense staining was observed in the stratified squamous epithelium of the forestomach. Staining was also observed in the esophagus, the tongue, the hard palate, and the skin but not in most other tissues including the columnar epithelium of the stomach or the lower gastrointestinal tract. GPI-PLD expression was also detected in several keratinocyte cell lines. Biochemical assays of glycosylphosphatidylinositol-degrading activity using [3H]myristate-labeled variant surface glycoprotein as substrate provided independent evidence for the presence of GPI-PLD. Expression of GPI-PLD by keratinocytes was not affected by culture in serum-free media, indicating that it does not originate by uptake of serum GPI-PLD in the media. These data suggest that keratinocytes are an important site of action of GPI-PLD and possibly a contributor to the plasma GPI-PLD pool.