Resistance to apoptosis is a prominent feature of malignant melanoma. Hyperthermic therapy can be an effective adjuvant treatment for some tumors including melanoma. We developed a fusion protein based on the tissue inhibitor of matrix metalloproteinase-1 linked to a glycosylphosphatidylinositol anchor (TIMP-1-GPI). The TIMP-1-GPI-fusion protein shows unique properties. Exogenous administration of TIMP-1-GPI can result in transient morphological changes to treated cells including modulation of proliferation and decreased resistance to apoptosis. The effect of TIMP-1-GPI on the biology of melanoma in the context of a defined hyperthermic dose was evaluated in vitro. Clonogenic assays were used to measure cell survival. Gelatinase zymography determined secretion of MMP-2 and MMP-9. Monoclonal antibody against FAS/CD95 was applied to induce apoptosis. The expression of pro- and anti-apoptotic proteins and the secretion of immunoregulatory cytokines were then evaluated using Western blot and ELISA. TIMP-1-GPI combined with a sub-lethal hyperthermic treatment (41.8 degrees C for 2 h) suppressed tumor cell growth capacity as measured by clonogenic assay. The co-treatment also significantly suppressed tumor cell proliferation, enhanced FAS receptor surface expression increased tumor cell susceptibility to FAS-mediated killing. The increased sensitivity to FAS-induced apoptosis was linked to alterations in the apoptotic mediators Bcl-2, Bax, Bcl-XL and Apaf-1. The agent works in concert with sub-lethal hyperthermic treatment to render melanoma cells sensitive to FAS killing. The targeted delivery of TIMP-1-GPI to tumor environments in the context of regional hyperthermic therapy could be optimized through the use of thermosensitive liposomes.