The effect of recombinant human tumor necrosis factor-alpha (rhTNF-alpha) on anti-tumor activity of adriamycin entrapped in small unilamellar liposomes (ADM-Lip) has been studied using BALB/c mice bearing subdermal Meth-A fibrosarcoma. Accumulation of i.v. injected ADM-Lip, but not of free ADM, into tumor tissue was augmented by a single i.v. injection of rhTNF-alpha. The maximum effect of rhTNF-alpha was expressed when it was injected 1 hr before ADM-Lip. This rhTNF-alpha treatment did not result in increased accumulation of ADM-Lip in normal organs. Based on these results, we treated mice bearing Meth-A tumor with rhTNF-alpha and then with ADM-Lip 1 hr later. By 2-cycle treatments using this regimen, tumor growth was markedly inhibited, and 5 out of 13 mice tested were cured of tumor, while treatment with rhTNF-alpha alone, ADM alone, ADM-Lip alone or rhTNF-alpha plus ADM resulted neither in prolongation of survival time nor in cure of mice. Our results indicate that ADM-Lip can express a potent and systemic anti-tumor activity after pretreatment of animals with TNF, but only when administration of these drugs is appropriately timed.