The activation of phospholipase D (PLD) by transforming Ras is well documented. Although two distinct PLD isoforms, PLD1 and PLD2, have been cloned from mammalian cells, it has remained unclear whether both isoenzymes are activated by Ras and, if this is the case, whether they are stimulated by a common mechanism. In the present study we show that expression of transforming Ras in HC11 mouse mammary epithelial cells enhanced the activity of endogenous PLD. Co-expression of Ras with either PLD1b or PLD2 resulted in elevated activities of both PLD isoenzymes in HC11 cells, indicating that transforming Ras was capable of activating both PLD isoforms in vivo. Ras-induced activation of PLD was resistant to the protein kinase C (PKC) inhibitor GF109203X, which preferentially affects conventional- and novel-type PKCs, but sensitive to Ro-31-8220, which inhibits atypical PKCs more effectively. Co-transfection of atypical PKC-ι with either PLD1b or PLD2 led to a selective activation of PLD2 by PKC-ι, whereas PLD1b was not affected. PLD1b, however, was found to be a potent activator of PKC-ι, whereas PLD2 was less effective in this respect. The data suggest that PKC-ι acts upstream of PLD2 and that PLD1b is implicated in the activation of PKC-ι. The data are discussed as indicating a putative signalling cascade comprising Ras → PLD1b →PKC-ι → PLD2. Evidence for the implication of this pathway in the transcriptional regulation of cyclin D1 is also presented.