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The uniqueness of Doxil can be attributed, to a large extent, to its intraliposomal doxorubicin-sulfate nanorod crystal. We re-examine these nanocrystal features and their mechanism of the formation by studying pegylated liposomal doxorubicins (PLDs) of the same lipid composition, size distribution, and extraliposome medium that were prepared at different ammonium sulfate (AS) concentrations. This study includes a comparison of the thermotropic behavior, morphology, and in vitro ammonia-induced doxorubicin release (relevant to Doxil's in vivo performance) of these PLDs. In this study, we confirm that a transmembrane ammonium gradient is critical for doxorubicin remote loading, and we demonstrate that the intraliposomal concentration of sulfate counteranions and ammonium ions determine to a large extent the physical state and stability of the PLDs' remote loaded doxorubicin. "Fully-developed" intraliposome doxorubicin-sulfate nanorod crystals (as defined by cryogenic transmission electron microscopy imaging) develop only when the ammonium sulfate (AS) concentration used for PLD preparation is ≥150 mM. Less than 10% of PLDs prepared with 100 mM AS show fully developed nanorod crystals. Intraliposomal AS concentration ≥200 mM is required to support the stable nanocrystallization in PLDs. The presence of nanocrystals and their melting enthalpy and phase transition co-operativity strongly affect the ammonia-induced doxorubicin release of PLDs. A quick, biphasic release occurs for PLDs that lack the nanorod crystals or have crystals of poor crystallinity, whereas PLDs prepared with ≥200 mM AS show a monophasic, zero-order slow release. This study also demonstrates that after remote loading, residual intraliposomal ammonium concentration and the transmembrane pH gradient related to it also play an important role in doxorubicin-sulfate intraliposomal crystallization and ammonia-induced doxorubicin release., Competing Interests: The authors declare the following competing financial interest(s): Yechezkel Barenholz is one of the inventors of two already expired (March 2010) patents relevant to Doxil: (1) Barenholz, Y., and Haran, G. “Method of Amphipathic Drug Loading in Liposomes by pH Gradient”. U.S. Patent 5,192,549, March 9, 1993. U.S. Patent 5,244,574, September 14, 1993; (2) Barenholz Y., and Haran, G. “Liposomes: Efficient Loading and Controlled Release of Amphipathic Molecules”, U.S. Patent 5,316,771, May 31, 1994. The Hebrew University received royalties from Doxil sales until the patents expired. The Barenholz Fund established with a portion of these royalties are used to support research in the Barenholz laboratory, including this study. Dima Shamrakov and Sioma Nudeman are employees of Ayana Ltd., a start-up founded by Yechezkel Barenholz and Yissum Hebrew University TTO. Ayana Ltd. works on the development of liposomal drugs including liposomal doxorubicin.