Interaction of natural killer (NK) cells with autologous immature dendritic cells (DCs) results in reciprocal activation; however, the underlying mechanisms are so far elusive. We show here that NK cells trigger immature DCs to polarize and secrete interleukin 18 (IL-18), a cytokine lacking a secretory leader sequence. This occurs through a Ca²⁺-dependent and tubulin-mediated recruitment of IL-18-containing secretory lysosomes toward the adhering NK cell. Lysosome exocytosis and IL-18 secretion are restricted at the synaptic cleft, thus allowing activation of the interacting NK cells without spreading of the cytokine. In turn, DC-activated NK cells secrete the proinflammatory cytokine high mobility group B1 (HMGB1), which induces DC maturation and protects DCs from lysis. Also HMGB1 is a leaderless cytokine that undergoes regulated secretion. Differently from IL-18, soluble HMGB1 is consistently detected in NK/DC supernatants. These data point to secretion of leaderless cytokines as a key event for the reciprocal activation of NK cells and DCs. DCs initiate NK cell activation by targeted delivery of IL-18, thus instructing NK cells in the absence of adaptive-type cytokines; in turn, activated NK cells release HMGB1, which promotes inflammation and induces DC maturation, thus favoring the onset of the adaptive immune response. (Blood. 2005;106:609-616)