Increases in intracellular Ca2+ concentration accompany many physiological events, including maturation of dendritic cells, professional antigen-presenting cells characterized by their ability to migrate to secondary lymphoid organs where they initiate primary immune responses. The mechanism and molecules involved in the early steps of Ca2+ release in dendritic cells have not yet been defined. Here we show that the concomitant activation of ryanodine receptor-induced Ca2+ release together with the activation of Toll-like receptors by suboptimal concentrations of microbial stimuli provide synergistic signals, resulting in dendritic cell maturation and stimulation of T cell functions. Furthermore, our results show that the initial intracellular signaling cascade activated by ryanodine receptors is different from that induced by activation of Toll-like receptors. We propose that under physiological conditions, especially when low suboptimal amounts of Toll-like receptor ligands are present, ryanodine receptor-mediated events cooperate in bringing about dendritic cell maturation.