Apparent binding affinity and capacity of daunomycin to the acidic phospholipids cardiolipin, phosphatidylglycerol, phosphatidic acid, phosphatidylserine and phosphatidylinositol, and the neutral zwitterion phosphatidylcholine were estimated via extraction of the drug with and without lipids into the organic phase of a biphasic organic-aqueous system (Folch) at various pH values. Total extraction was highest at pH 8.50 (approximately 70% of total drug at 1 mumole/3.6 mL concentration), medium at pH 2.77 (approximately 50%) and lowest at pH 6.55 (approximately 20%). Extraction was highly specific in the presence of all phospholipids except phosphatidylcholine at pH 6.55, and specific for cardiolipin and phosphatidylglycerol at pH 2.77. However, there was no phospholipid-specific extraction at pH 8.50. Apparent binding affinity decreased in the order phosphatidic acid much greater than phosphatidylserine greater than phosphatidylinositol greater than phosphatidylglycerol much greater than cardiolipin. No specific extraction was detectable in the presence of the zwitterionic phosphatidylcholine. However, cardiolipin was the only phospholipid with two binding sites per daunomycin molecule, whereas all others have only one binding site per daunomycin molecule.