Rat brain cytosol contains proteins that markedly inhibit the activity of partially purified brain membrane phospholipase D (PLD) stimulated by ADP-ribosylation factor (Arf) and phosphatidylinositol 4,5-bisphosphate (PIP2). Sequential chromatography of the brain cytosol yielded four inhibitor fractions, which exhibited different kinetics to heat treatment at 70°C. Purification of the most heat-labile inhibitor to homogeneity yielded two preparations, which displayed apparent molecular masses of 150 kDa and 135 kDa, respectively, on SDS-polyacrylamide gels. Tryptic digests of the 150- and 135-kDa proteins yielded similar elution profiles on a C18 reverse-phase column, suggesting that the 135-kDa form is a truncated form of the 150-kDa form. Sequences of two tryptic peptides were determined. A data base search revealed no proteins with these sequences. The purified 150-kDa inhibitor negated the PLD activity stimulated by Arf, RhoA, or Cdc42. The concentration required for half-maximal inhibition was 0.4 nM. Concentration dependence on the 150-kDa inhibitor was not affected by changes in the concentrations of Arf, PIP2, or phosphatidylcholine used in the assays, suggesting that the inhibition is not due to competition with the activators or substrate for PLD. The purified inhibitor did not affect the PIP2-hydrolyzing activity of a phospholipase C isozyme that was measured with substrate vesicles of lipid composition identical with that used for the PLD assay. Thus, the mechanism of inhibition appears to be a specific allosteric modification of PLD rather than disruption of substrate vesicles.