Abstract Actinomycin D was encapsulated within liposomes in two different ways. When the drug was incorporated in the lipid phase, as part of the membrane bi-layers of the liposomes, they were called “lipid phase liposomes” (LPL); when the drug was incorporated in the aqueous solution in the center and between the lipid bi-layers of the liposomes, they were called “aqueous phase liposomes” (APL). Distributions of 3H-actinomycin D in tissues of mice were determined from 15 minutes to 48 hours after a single intravenous injection of either LPL, APL, or nonencapsulated actinomycin D. Marked differences in tissue distribution were shown. 3 H-actinomycin D incorporated in LPL showed high concentrations in the lungs and low concentrations in the intestinal wall; whereas the reverse was found with APL. The spleen and bone marrow of mice receiving LPL showed an increase in 3 H-radioactivity between 3 and 24 hr after injection that was closely correlated with a concomitant decrease in activity in the liver and lungs. Mice receiving either APL or nonencapsulated actinomycin D had higher levels of the drug in the blood, kidneys and intestinal wall than did mice receiving LPL. In vitro studies showed that 3 H-actinomycin D leaked out from APL significantly faster than that from LPL. We have demonstrated that the tissue distribution of a drug can be modified not only by liposome encapsulation, but also by varying the way of incorporating the drug within liposomes, and thereby altering the surface properties. The two forms of incorporating drugs within liposomes are potentially useful to direct antitumor agents to specific tumor bearing tissues.