High-pressure Fourier-transform infrared (FT-IR) spectroscopy was used to study the barotropic behaviour of phosphatidylserine bilayers and their interactions with the local anesthetic tetracaine. The model membrane systems studied were multilamellar aqueous dispersions of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) in the absence and the presence of tetracaine at pH 5.5 and 9.5. The infrared spectra were measured at 28ºC in a diamond anvil cell as a function of pressure up to 25 kbar. The results show that the barotropic behavior of the negatively charged phosphatidylserine bilayers is very similar to that observed for zwitterionic phosphilipids, such as phosphatidylcholine and phosphatidylethanolamine, with corresponding acyl chains The results also indicate that the local anesthetic partitions into phosphatidylserine bilayers in an environment close to the membrane-water interface and interacts electrostatically with the lipid head group. Application of high hydrostatic pressure on the lipid-anesthetic systems results in the pressure-induced expulsion of the anesthetic from a membrane to an aqueous environment. The pressures prequired for expulsion of anesthetic from bilayers are much higher for the unsaturated lipid (DOPS) than for the saturated lipid (DMPS) (≈6 kbar vs ≈2 kbar, respectively). Whereas incorporation of the anesthetic into DOPS bilayers does not affect significantly the structural and dynamic properties of the disordered acyl chains in the liquid-crystalline phase, it orders the DMPS acyl chains in the gel phase.