An intravenously injectable sustained-release delivery system for mitomycin C (MMC) was prepared by formulating a lipophilic MMC prodrug, N-(cholesteryloxycarbonyl)glycyl MMC (COCG-MMC), in unilamellar liposomes, and its blood disposition and antitumor activity were investigated in mice. Liposomes composed of egg phosphatidylcholine, egg sphingomyelin and COCG-MMC in a molar ratio of 7 : 3 : X (X=0-2.7) were prepared by the combination of controlled dialysis or sonication and sequential extrusion. All the preparations prepared by controlled dialysis showed a fairly narrow size distribution. After intavenous injection of COCG-MMC-bearing liposomes at a dose of 20 μmol/kg as a prodrug, the blood levels of regenerated MMC were maintained for the first 7 h in the range of 1.16 to 0.48 μM. In contrast, when an equimolar amount of MMC was given in an aqueous solution, MMC was rapidly cleared with little remaining in the circulation after 2 h. COCG-MMC exhibited significant cytotoxicity against P 388 leukemia in vitro and its activity was approximately one-third that of the parent drug. Prodrug-bearing liposomes inhibited the growth of subcutaneously-implanted Colon 26 adenocarcinoma and human mammary carcinoma MX-1 xenograft. Compared with MMC aqueous solution, prodrug-bearing liposomes showed reduced antitumor activity and reduced toxicity. In each tumor system, the body weight change differences (test minus controls), indices of host toxicity, at the ID₅₀'s (the doses which inhibit tumor growth to 50% of controls) showed no significant difference between these dosage forms. The results indicate that COCG-MMC-bearing liposomes successully maintain blood MMC levels over a prolonged period of time, but their therapeutic efficacy is almost equal to that of MMC aqueous solution.