We describe a quantitative model for assessing the cytolytic activity of antigen-specific CD8⁺ T cells in vitro and in vivo in which the concentration of antigen-specific CD8⁺ T cells determines the efficiency with which these cells kill cognate antigen–expressing melanoma cells in packed cell pellets, in three-dimensional collagen-fibrin gels in vitro, and in established melanomas in vivo. In combination with a clonogenic assay for melanoma cells, collagen-fibrin gels are 4,500–5,500-fold more sensitive than the packed cell pellet–type assays generally used to measure CD8⁺ T cell cytolytic activity. An equation previously used to describe neutrophil bactericidal activity in vitro and in vivo also describes antigen-specific CD8⁺ T cell–mediated cytolysis of cognate antigen-expressing melanoma cells in collagen-fibrin gels in vitro and in transplanted tumors in vivo. We have used this equation to calculate the critical concentration of antigen-specific CD8⁺ T cells, which is the concentration of these cells required to hold constant the concentration of a growing population of cognate antigen-expressing melanoma cells. It is ∼3.5 × 10⁵/ml collagen-fibrin gel in vitro and ∼3 × 10⁶/ml or /g melanoma for previously published studies of ex vivo–activated adoptively transferred tumor antigen–specific CD8⁺ T cell killing of cognate antigen–expressing melanoma cells in established tumors in vivo. The antigen-specific CD8⁺ T cell concentration required to kill 100% of 2 × 10⁷/ml cognate antigen-expressing melanoma cells in collagen fibrin gels is ≥10⁷/ml of gel.