Autoclaving of liposomes composed of egg phospholipids or saturated phospholipids, the latter sometimes combined with cholesterol, was performed in an isotonic acetate buffer (pH 4.0) or Hepes buffer (pH 7.4). After a standard autoclaving cycle (15 min, 121°C), no change could be observed in pH, size, and extent of oxidation. Dependent on the experimental conditions, a minor or substantial increase in the fraction of hydrolyzed phospholipids was found. After a sterilization cycle, pronounced leakage was found for a water-soluble, encapsulated compound (calcein) and for an amphiphilic compound (doxorubicin). Lipophilic, liposome bilayer-associated compounds [N-trifluoroacetyldoxorubicin-14-valerate (AD-32) and α-tocopherol] remained in the liposomes after autoclaving. However, substantial degradation of AD-32 was observed. Under proper conditions liposomes without or with thermostable, lipophilic drugs can be sterilized by autoclaving. However, the hydrolysis of phospholipids can pose a problem, as hydrolysis kinetics depend on the pH used. In the chosen circumstances the autoclaving cycle caused massive loss of hydrophilic, nonbilayer interacting compounds; under those conditions “free” drug removal or drug encapsulation should be performed after the autoclaving step.