In a wide variety of cells, phosphatidylcholine hydrolysis in response to diverse agents is catalyzed by phospholipase D (PLD) activities that are believed to be membrane-bound. Indeed, PLD has been detected in membrane fractions of several tissues and cells. We now demonstrate in various bovine tissue including lung, brain, spleen, heart, kidney, thymus, and liver as well as rat lung that a great majority of the detectable PLD activity is cytosolic. This cytosolic PLD activity differs from a less abundant membrane-bound isozyme by chromatographic mobilities on anion exchange and gel filtration columns, by substrate specificity, by substrate concentration dependence, and by divalent cation and detergent effects. Fractionation of the cytosol by anion exchange chromatography enhances PLD activity up to 20-fold, suggesting the presence in the cytosol of PLD inhibitory factor(s). We conclude that mammalian PLD exists in multiple forms and that appropriate selection of assay conditions is critical for observing PLD activity in the cytosol.