To study the conditions for preparing liposomes with a high internal protein content the uptake of creatine phosphokinase by the liposomes was studied quantitatively under different experimental conditions. Addition of 20% of a positively charged lipid (stearylamine) to the lipid mixture followed by ultrasonic treatment of the liposomes for 1 min gave the highest levels of latent activity of the enzyme. Maximal uptake (6.1%) was obtained by the use of a 300 mM glucose solution, pH 7.0, containing 0.5 mg protein/ml, as the aqueous phase. Washing the preparations of liposomes with physiological saline at 205,000g gave minimal external enzyme activity. When the liposomes were kept for 7 days under argon protection at 4°C there was no decrease in the internal protein content and no increase in external activity.