A new derivative of L-asparaginase, palmitoyl-L-asparaginase (palmitoyl-L-ASNase), has been incorporated in liposomes. For this purpose we modified the dehydration-rehydration method and optimized the liposomal composition. The pharmacokinetics, toxicity, and in vivo antitumor activity against P1534 lymphoma of different liposomal palmitoyl-L-ASNase formulations were studied. Liposomal encapsulation of palmitoyl-L-ASNase as compared with free palmitoyl-L-ASNase resulted in a prolongation of the blood half-life (from 2.88 h to longer than 23.7 h), abrogation of acute toxicity, and preservation of in vivo antitumor activity.