Adenoviral vectors expressing tumor-associated antigens can be used to evoke a specific immune response and inhibit tumor growth. In this study, we tested the efficacy of adenoviral vectors encoding human gp100 (Ad2/hugp100), murine gp100 (Ad2/mugp100), or murine TRP-2 (Ad2/muTRP-2) for their ability to elicit a specific cellular immune response and inhibit the growth of B16 melanoma tumor cells in the mouse. C57BL/6 mice were immunized with Ad2/hugp100, Ad2/mugp100, or Ad2/muTRP-2 either 2 weeks prior to B16-F10 tumor challenge (prophylactic treatment) or 3 days after tumor challenge (active treatment). Ad2/hugp100 and Ad2/muTRP-2 administered to two or more intradermal (i.d.) sites inhibited subsequent subcutaneous tumor growth in > or = 80% of the mice and elicited an antigen-specific cytotoxic T lymphocyte response, whereas other administration routes were not as effective. Ad2/mugp100 administered to two i.d. sites did not inhibit tumor growth or provoke cellular immunity. Immunization was less effective with active treatment where tumor growth was not significantly inhibited by a single dose of either Ad2/muTRP-2 or Ad2/hugp100. However, increasing the number of intradermal immunization sites and the number of doses resulted in progressive improvements in protection from tumor growth in the active treatment model. In conclusion, breaking host tolerance to elicit protective immunity by using adenoviral vectors expressing melanoma-associated antigens is dependent upon the choice of antigen, the site of administration, and the number of doses. These observations provide insights into the clinical applicability of adenoviral vaccines for immunotherapy of malignant diseases.