A large number of pharmaceutically active compounds have a high affinity to acidic phospholipids; good examples are the cationic compounds lidocaine, propranolol, and gentamycin. These drugs influenced the lipid dynamics of liposomes composed of phosphatidylcholine and the acidic phosphatidylglycerol, as judged by the excimer/monomer emission intensity ratio for a pyrene-labeled phospholipid analog, as well as by polarization of DPH fluorescence. When the mole fraction X of PG (XPG) was 0.20, lidocaine increased membrane fluidity. The opposite was true for propranolol, which caused the formation of pyrene lipid-enriched microdomains. Gentamycin had no apparent effect. At XPG = 1.00, all these drugs rigidified membrane. Subsequently, we investigated the detachment of a cationic peripheral membrane protein, cytochrome c (cyt c), by these compounds from liposomes. This was accomplished by monitoring resonance energy transfer from a pyrene-labeled phospholipid to the heme of cyt c. The efficiency of the above compounds to dissociate cyt c varied considerably. In brief, significantly lower concentrations of gentamycin than propranolol or lidocaine were required for half-maximal dissociation of cyt c from liposomes, although the final extent of protein detachment by gentamycin was less complete. ATP augmented the dissociation of cyt c from membranes by lidocaine and propranolol. Stopped-flow measurements also revealed that the half-times differed for the release of cyt c from the membranes. Our results are likely to reflect differences in the contributions of the electrostatic interactions and hydrophobicity to the drug/lipid interaction and comply with two different acidic phospholipid binding sites in cyt c.