Cellular adhesion can be regulated by, as yet, poorly defined intracellular signalling events. Phospholipase D enzymes generate the messenger lipid phosphatidate and here we demonstrate that suppression of this reaction inhibits cellular adhesion. This effect was reversed by the addition of cell-permeable analogues of either phosphatidate or phosphatidylinositol 4,5-bisphosphate. By contrast, neither diacylglycerol nor lysophosphatidic acid were able to reverse this effect suggesting that phosphatidate itself acts directly on a target protein(s) to regulate adhesion rather than as the result of its conversion to either of these metabolite lipids. Antibodies that block beta1 and beta2 integrin-substrate interactions inhibited adhesion stimulated by both phosphatidate and phosphatidylinositol 4,5-bisphosphate indicating that these lipids regulate beta1 and beta2 integrin-mediated adhesion. In vivo, these lipids can be generated by phospholipase D2 and phosphatidylinositol 4-phosphate 5-kinase Igamma b, respectively, and over-expression of catalytically-functional forms of these enzymes dose-dependently stimulated adhesion while siRNA depletion of PLD2 levels inhibited adhesion. Furthermore the ability of over-expressed phospholipase D2 to stimulate adhesion was inhibited by a dominant-negative version of phosphatidylinositol 4-phosphate 5-kinase Igamma b. Consistent with this, phosphatidylinositol 4-phosphate 5-kinase Igamma b-mediated adhesion was dependent upon phospholipase D2's product, phosphatidate indicating that phosphatidylinositol 4-phosphate 5-kinase Igamma b is downstream of, and necessary for, phospholipase D2's regulation of adhesion. It is likely that this phospholipase D2-generated phosphatidate directly stimulates phosphatidylinositol 4-phosphate 5-kinase Igamma b to generate phosphatidylinositol 4,5-bisphosphate as this mechanism has previously been demonstrated in vitro. Thus, our data indicates that during the initial stages of adhesion, phospholipase D2-derived phosphatidate stimulates phosphatidylinositol 4-phosphate 5-kinase Igamma b to generate phosphatidylinositol 4,5-bisphosphate and that consequently this inositol phospholipid promotes adhesion through its regulation of cell-surface integrins.