Mammalian phospholipase D (PLD) has been implicated in the cellular signal transduction pathways leading to diverse physiological events and known to be regulated by many cellular factors. To identify the proteins that interact with PLD, we performed a protein overlay assay with fractions obtained from the sequential column chromatographic separation of rat brain cytosol using purified PLD2 as a probe. A protein of molecular mass 40 kDa, which was detected by anti-PLD antibody with overlaying of the purified PLD2, is shown to be aldolase C by peptide-mass fingerprinting with matrix-assisted laser desorption/ionization-time-of flight mass spectrometry (MALDI-TOF-MS). Aldolase A also showed similar binding properties as aldolase C and was co-immunoprecipitated with PLD2 in COS-7 cells overexpressing PLD2 and aldolase A. The PH domain corresponding to amino acids 201-310 of PLD2 was necessary for the interaction observed in vitro, and aldolase A was found to interact with the PH domain of PLD2 specifically, but not with other PH domains. PLD2 activity was inhibited by the presence of purified aldolase A in a dose-dependent manner, and the inhibition by 50% was observed by the addition of less than micromolar aldolase A. Moreover, the inclusion of the aldolase metabolites fructose 1,6-bisphosphate (F-1,6-P) or glyceraldehyde 3-phosphate (G-3-P) resulted in an enhanced interaction between PLD2 and aldolase A with a concomitant increase in the potential ability of aldolase A to inhibit PLD2, which suggests the existence of a possible regulation of the interaction by the change of intracellular concentrations of glycolytic metabolites.