Antigen-specific priming of human, naive T cells has been difficult to assess. Owing to the low initial frequency in the naive cell pool of specific T cell precursors, such an analysis has been obscured by the requirements for repeated stimulations and prolonged culture time. In this protocol, we describe how to evaluate antigen-specific priming of CD8+ cells 10 d after a single specific stimulation. The assay provides reference conditions, which result in the expansion of a substantial population of antigen-specific T cells from the naive repertoire. Various conditions and modifications during the priming process (eg, testing new cytokines, co-stimulators and so on) can now be directly compared with the reference conditions. Factors relevant to achieving effective priming include the dendritic cell preparation, the T cell preparation, the cell ratio at the time of priming, the serum source used for the experiment and the timing of addition and concentration of the cytokines used for expansion. This protocol is relevant for human immunology, vaccine biology and drug development.