Dendritic cells (DC), in their role in initiation of the adaptive immune response, have been extensively studied for their capacity to interact and stimulate naive T cells. Subsets of mature murine DC isolated directly from the spleen have been shown to differ in their ability to induce proliferative responses in both primary CD4(+) and primary CD8(+) T cells; the myeloid-related CD8alpha(-) DC induce a more intense or prolonged proliferation of naive T cells than do the lymphoid-related DC bearing CD8alpha despite similar expression of MHC and co-stimulatory molecules. Here we examine the interaction of these DC subpopulations with T cells already in the activated or memory state which are known to have greater sensitivity to antigen stimulation and bear receptors with increased capacity for signal transduction. We show that influenza virus-specific CD4(+) T cell clones and splenic T cells from peptide-primed animals proliferated in response to antigen presented by separated splenic CD8(-) DC. In contrast, these T cells showed only weak, if any, proliferation in response to CD8(+) DC despite observable cluster formation in the cultures. The differential between the two DC types in inducing proliferation was even more pronounced than previously seen with primary T cells and did not reflect differential longevity of the DC in culture, altered response kinetics or deviation from IL-2 to IL-4 induction with CD8(+) DC, but was related to the levels of IL-2 induced. The deficiency in the CD8(+) DC was not overcome by using infectious virus rather than synthetic peptide as the antigen source. These results show that lymphoid-related CD8(+) splenic DC, despite their mature phenotype, fail to provide appropriate signals to secondary CD4(+) T cells to sustain their proliferation.