The association of phospholipase (PLD)-1 with protein kinase C-related protein kinases, PKNalpha and PKNbeta, was analyzed. PLD1 interacted with PKNalpha and PKNbeta in COS-7 cells transiently transfected with PLD1 and PKNalpha or PKNbeta expression constructs. The interactions between endogenous PLD1 and PKNalpha or PKNbeta were confirmed by co-immunoprecipitation from mammalian cells. In vitro binding studies using the deletion mutants of PLD1 indicated that PKNalpha directly bound to residues 228-598 of PLD1 and that PKNbeta interacted with residues 1-228 and 228-598 of PLD1. PKNalpha stimulated the activity of PLD1 in the presence of phosphatidylinositol 4,5-bisphosphate in vitro, whereas PKNbeta had a modest effect on the stimulation of PLD1 activity. The stimulation of PLD1 activity by PKNalpha was slightly enhanced by the addition of arachidonic acid. These results suggest that the PKN family functions as a novel intracellular player of PLD1 signaling pathway.