Hexadecylphosphorylcholine (HePC, D-18506, INN: Mitelfosine) belongs to the family of alkylphosphocholines with anticancer activity. Previous reports have related its antitumoral activity to their ability to interfere with phospholipid metabolism. However a clear mechanism of action has not been established yet. We have investigated the effect of HePC on two enzymes recently reported to play a role in cell growth proliferation, phospholipase D (PLD) and choline kinase (ChoK). Our results demonstrate that treatment with HePC induces a rapid stimulation of PLD, that may be achieved by PKC dependent or independent mechanisms, depending on the cell line investigated. Both PLD1 and PLD2 isoenzymes are sensitive to HePC activation. By contrast, no effect was observed by HePC on ChoK, a new target for anticancer drug development. Furthermore, in all cell lines tested, a chronic exposure of the cells to HePC abrogates PLD activation by either phorbol esters or HePC itself with no effect on total cellular PLD levels. This is reflected in a strong inhibition of PLD activity. We suggest that the inhibitory effects on PLD by HePC may be related to its antitumoral action.