The conventional enzyme-linked immunospot (ELISPOT) technique detects only one secreted cytokine, which constitutes a major pitfall for the accurate characterization of the various T-cell subpopulations. We have therefore developed a fluorospot assay, which is a modification of the ELISPOT and is based on the use of multiple fluorescent-labeled anticytokines detection antibodies. A special automated ELISPOT reader consisting of a light microscope with incident fluorescence illumination and an integrating digital color camera has been adapted for this technique. This technique has been applied for the analysis of subpopulations of T-cells and polarized antigen-specific T-cells.