T cell activation by APC requires cytosolic Ca 2+([Ca 2+] i) elevation. Using two-photon microscopy, we visualized Ca 2+ signaling and motility of murine CD4+ T cells within lymph node (LN) explants under control, inflammatory, and immunizing conditions. Without Ag under basal noninflammatory conditions, T cells showed infrequent Ca 2+ spikes associated with sustained slowing. Inflammation reduced velocities and Ca 2+ spiking in the absence of specific Ag. During early Ag encounter, most T cells engaged Ag-presenting dendritic cells in clusters, and showed increased Ca 2+ spike frequency and elevated basal [Ca 2+] i. These Ca 2+ signals persisted for hours, irrespective of whether T cells were in contact with visualized dendritic cells. We propose that sustained increases in basal [Ca 2+] i and spiking frequency constitute a Ca 2+ signaling modality that, integrated over hours, distinguishes immunogenic from basal state in the native lymphoid environment.