A critical characteristic of optimized liposomal delivery systems is controlled drug release. This report describes the use of the monoacyl cationic lipids stearylamine (SA) and sphingosine (SPH), in conjunction with a transmembrane pH gradient, to enhance retention of the anticancer agent vincristine within liposomes administered intravenously to mice. The addition of SA to liposomes composed of distearoylphosphatidylcholine (DSPC) and cholesterol (Chol), to achieve a final composition of DSPC/Chol/SA at ratios of 45/45/10 (mol/mol/mol), caused a significant improvement in vincristine retention in the circulation of mice when combined with a transmembrane pH gradient of 5 units. Specifically, the presence of SA in liposomes having an internal pH (pHi) of 2.0 had a plasma vincristine AUC of 41.8 mg.h/100 mL plasma, compared to a vincristine AUC in the plasma of 28.0 mg.h/100 mL for identical liposomes lacking SA; an increase of 49%. Similar effects were observed with 10 mol% of the cationic lipid SPH. Comparison to control formulations lacking SA or SPH, or with smaller pH gradients (3 units), showed that improved vincristine retention required both the cationic lipid and the larger pH gradient. The presence of SA or SPH significantly improved vincristine pharmacokinetics due to the increased drug retention, but did not adversely alter the liposome pharmacokinetics. Enhanced retention of vincristine using SPH and a transmembrane pH gradient was associated with significant increases in anti-tumor activity against the murine P388 leukemia. The results are directly relevant to the use of charged lipid components to improve the drug retention/release attributes and the use of cationic lipids to promote accumulation at tumor-associated vascular endothelium.