It has been observed that the efficient transfection of T cells by RNA electroporation requires prior activation of T cells with mitogens or by anti-CD3 antibody stimulation. We hypothesized that this requirement for T cell activation could be leveraged to express marker genes within activated T cells responding to antigen-pulsed dendritic cells and allow for the selective enrichment and modification of antigen-specific T cells. Using electroporation of mRNA encoding green fluorescent protein as a marker gene, we demonstrate that RNA electroporation can efficiently allow for the separation of cytomegalovirus-specific CD8+ and CD4+ T cells from bulk culture responding to cytomegalovirus pp65 antigen-pulsed dendritic cells. Furthermore, we demonstrate that cytomegalovirus-specific T cells can be functionally modified by RNA transfection of the C-X-C chemokine receptor, CXCR2, to migrate efficiently toward a variety of CXCR2-specific chemokines in vitro and in vivo. These studies demonstrate the utility of RNA transfection as a simple method by which to purify and selectively modify the function of antigen-specific T cells for use in adoptive immunotherapy, and importantly provide evidence that transient expression of proteins by RNA transfection is an efficient means of modulating the in vivo function of activated T cells.