To characterize the replicative capacity of human central memory (T(CM)) CD4 T cells, we have developed a defined culture system optimized for the ex vivo expansion of Ag-specific CD4(+) T cells. Artificial APCs (aAPCs) consisting of magnetic beads coated with Abs to HLA class II and a costimulatory Ab to CD28 were prepared; peptide-charged HLA class II tetramers were then loaded on the beads to provide Ag specificity. Influenza-specific DR*0401 CD4 T(CM) were isolated from the peripheral blood of normal donors by flow cytometry. Peptide-loaded aAPC were not sufficient to induce resting CD4 T(CM) to proliferate. In contrast, we found that the beads efficiently promoted the growth of previously activated CD4 T(CM) cells, yielding cultures with >80% Ag-specific CD4 cells after two stimulations. Further stimulation with peptide-loaded aAPC increased purity to >99% Ag-specific T cells. After in vitro culture for 3-12 wk, the flu-specific CD4 T(CM) had surface markers that were generally consistent with an effector phenotype described for CD8 T cells, except for the maintenance of CD28 expression. The T(CM) were capable of 20-40 mean population doublings in vitro, and the expanded cells produced IFN-gamma, IL-2, and TNF-alpha in response to Ag, and a subset of cells also secreted IL-4 with PMA/ionomycin treatment. In conclusion, aAPCs expand T(CM) that have extensive replicative capacity, and have potential applications in adoptive immunotherapy as well as for studying the biology of human MHC class II-restricted T cells.