Methotrexate is a cell-cycle phase-specific antimetabolite that requires prolonged exposure for optimal efficacy. However, the in vivo half-life is short, and repeated administrations are required for optimal efficacy. Methotrexate was encapsulated into a lipid-based drug-delivery system to create an extended-release formulation (Depo/methotrexate) for subcutaneous administration. Pharmacokinetics and efficacy studies were done in BDF1 mice. Depo/methotrexate increased the methotrexate plasma half-life by a factor of 190, from 0.53 to 100 h. Plasma peak levels of the encapsulated drug were 120-fold lower than those of unencapsulated methotrexate, whereas the AUCs (areas under the concentration-time curve) were similar. As a consequence of the extended drug release, the single-dose potency of methotrexate against the L1210 leukemia model was increased by a factor of about 130 without producing significant changes in the therapeutic index. In conclusion, Depo/methotrexate appears to have potential usefulness as an extended-release formulation of methotrexate.