As a part of our ongoing search for a safe and efficient anti-tumor vaccine, we attempted to determine whether the molecular nature of certain tumor antigens would influence immune responses against tumor cells. As compared with freeze-thawed or formaldehyde-fixed tumor antigens, heat-denatured tumor antigens elicited profound anti-tumor immune responses and greatly inhibited the growth of live tumor cells. The heat-denatured tumor antigens induced a substantial increase in the anti-tumor CTL response in the absence of any adjuvant material. This response appears to be initiated by strong activation of the antigen-presenting cells, which may recognize heat-denatured protein antigens. Upon recognition of the heat-denatured tumor antigens, macrophages and dendritic cells were found to acutely upregulate the expression of co-stimulatory molecules such as B7.2, as well as the secretion of inflammatory cytokines such as IL-12 and TNF-alpha. The results of this study indicate that heat-denatured tumor extracts might elicit protective anti-tumor adaptive immune responses and also raise the possibility that a safe and efficient adjuvant-free tumor vaccine might be developed in conjunction with a dendritic cell-based tumor vaccine.