Triggering of the T-cell receptor by cognate antigen induces a variety of cellular events leading to cell proliferation and differentiation. While the plasticity and diversity of T-cell responses have been recognized for a long time, few quantitative studies have been conducted to measure what proportion of specific T cells will enter a given differentiation program after antigen stimulation. In the present study, we analyzed human T cells cultured with influenza-peptide-loaded dendritic cells. We compared three individual methods for assaying the frequency of antigen-specific T cells: ELISPOT, tetramer-binding, and proliferation. The three methods yielded similar but not identical results. In order to study these differences at the single cell level, we developed a multiparameter flow cytometric method, which allows simultaneous analysis of antigen-specific tetramer binding, T-cell proliferation, and cytokine production. Based on these data, we used flow precursor frequency analysis to calculate the proportion of eight different precursor subsets in the original, resting population. We conclude that approximately half of the cells that bound specific tetramers actually proliferated and synthesized IFNgamma in response to antigen. In addition, similar numbers of cells that did not bind tetramer proliferated (but did not synthesize IFNgamma). The method allows for an estimate of the precursor frequency of each functional subset within the initial population. It could be applied to additional markers of function and differentiation, combining all parameters into a description of the complex response potential of a T-cell pool.