It has been reported that mammalian serum, and to a lower extent mammalian liver, brain, pancreas, udder, and milk, contain glycosylphosphatidylinositol-specific phospholipase D activity. However, the sites of synthesis have not been determined. In order to study in which cell(s) of the organism synthesis of glycosylphosphatidylinositol-specific phospholipase D takes place, we undertook a systematic screening of 12 different bovine tissues. In situ hybridization experiments with a specific anti-sense RNA probe, derived from a bovine liver cDNA, revealed that glycosylphosphatidylinositol-specific phospholipase D mRNA is present in mast cells of the adrenal gland, lung, and liver. On the other hand, our specific probe detected no mRNA in bovine pancreas, brain, and udder, although enzyme activity has been reported in these tissues. Northern blot analysis of total bovine liver RNA demonstrated two distinct glycosylphosphatidylinositol-specific phospholipase D mRNAs of approximately 3.3 kb and 4 kb length suggesting that two forms of the enzyme may exist.