Phospholipase D(PLD) is the enzyme catalyzing the hydrolysis of the terminal phosphcester bond of phospholipid head group to produce phosphatidic acid and the corresponding base. The effect of spermine on the PLD activity of rat brain mitochondrial preparation was investigated. Spermine, in the presence of oleic acid, activates the rat brain mitochondrial PLD, whose effect was further enhanced by the presence of divalent cation, $Ca^{2+}$, $Mg^{2+}$, and $Ba^{2+}$. Among the various monoamines tested, only histamine at the high concentration was effective in activation the PLD. Polylysine increased the PLD activity, particularly, the longer chain of the molecule activated the PLD more effectively. There was no significant difference in the substrate specificity for the PLD activity between phosphatidylcholine(PC) and phoshpatidylethanolamine (PE). This substrate specifitiy is different from the PE specificity reported for the intestinal mitochondrial PLD.