CD8(+) T lymphocytes are capable of recognizing and destroying cancer cells or virally infected cells and can thus offer protection from cellular malignant transformation and pathogenic challenges. With large numbers of genes discovered in genome analyses, rapid identification of cancer or viral antigens would facilitate better exploitation of CD8(+) T lymphocyte-mediated immune protection. Reverse transfection microarray technology allows expression of individual cDNAs at defined positions in a cell monolayer and direct detection of corresponding phenotypic changes of transfected cells at specific locations. In this study, we have integrated reverse transfection with image-based fluorometric detection of antigen-specific CTL-mediated cytotoxicity on transfected cell microarrays. As a result, the antigen recognition of cloned CTL cells has been successfully detected on the cell microarray, in which the specific or non-specific mini-gene DNA in the expression vector or vector alone was spotted. Moreover, the cellular capability of antigen processing and presentation on microarray has also been evaluated by using chimeric DNA constructs containing the antigen-encoding mini-gene sequence. This novel approach may facilitate high throughput screens of cancer cell or virus cDNA libraries to identify individual cDNAs that encode targets for immune intervention.