Multilamellar liposomes loaded with D-cycloserine (D-CS) were prepared by a thin layer evaporation technique, followed by freezing and thawing cycles. Charged components and bioadhesive material, such as distearolylphosphatitylethanolamine covalently coupled with methoxypolyethyleneglycol, were used to prepare liposomes with different physico-chemical and technological properties. Negatively charged liposomes showed higher D-CS encapsulation efficiency (about 37%, w/w) than neutral and positively charged liposomes (about 5 and 17%, w/w, respectively). All formulations showed in vitro, after a burst effect, a prolonged release of the encapsulated drug. Lipid vesicles made of dipalmitoylphosphatidylcholine (DPPC) were used as a biomembrane model to evaluate in vitro the interaction of D-CS with biological membranes. Differential scanning calorimetry was used as a simple and noninvasive technique of analysis. D-CS was distributed in the aqueous compartments of liposomes for interaction with the phospholipid polar head-groups (enhancement of Delta H value). However, due to its high diffusibility the drug was also able to freely permeate through DPPC liposomes, altering during this passage the hydrophobic domains of the bilayers. Stability studies were performed at different temperatures and pH values to assay the integrity of the drug during the liposome production steps. D-CS was rapidly degraded at acidic pH, but no significant hydrolysis was observed at pH 7.4 after 7 days.