Abstract Phospholipase D (PLD) is regulated by many factors, including protein kinase C (PKC) and small G-proteins of the Rho and ADP-ribosylation factor families. Previous studies revealed that the activation of PLD1 by phorbol ester is associated with the binding of PKCα to a site in the N-terminus of PLD1. The purpose of the present study was to determine this site more precisely. Immunoprecipitation with a series of four PLD1 deletion mutants confirmed that PKCα strongly interacted with the amino acid sequence 1–318 at the N-terminus of PLD1 and weakly with the sequence 841–1036 at the C-terminus. Further immunoprecipitation studies with deletion mutants of the 1–318 and 1–215 PLD1 fragments revealed that there were binding sites in the 1–49 N-terminal sequence and also in the 216–318 sequence containing the PH domain. Studies of N-terminal deletion mutants of full-length PLD1 confirmed the presence of a binding site in the 1–49 sequence and a further site in the 1–318 sequence. Both deletion mutants showed impaired activation by PKCα in vivo, but unchanged activation by active V 14RhoA. These findings identify the 1–49 sequence is a major binding/activation site for PKCα on PLD1, but also indicate involvement of the PH domain.