We studied the effects of various phoapholipids on the DNA synthesizing reactions by calf thymus DNA polymerases α, δ, and ε. Of these three enzymes, DNA polymerase ε was most sensitive to acidic phospholipids, i.e., phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), phosphatidylserine (PS), phosphatidic acid (PA), and cardiolipin (CAR). Of these acidic phospholipids, PI (from bovine liver) is of special interest because it inhibited DNA polymerase ε much more strongly than DNA polymerase α and δ. The inhibition of DNA polymerase ε by PI was competitive with the DNA template-primer and was noncompetitive with dTTP substrate. The K1 value was estimated to be 16 μM. These results indicate that PI from bovine liver can be used as a specific inhibitor for DNA polymerase ε to analyze its role in DNA replication. Interestingly, the PI isolated from soybean, which has a different fatty acid composition, inhibited not only DNA polymerase ε but also DNA polymerase α.