Kupfer-type immunological synapses are thought to mediate intercellular communication between antiviral T cells and virally infected target Ag-presenting brain cells in vivo during an antiviral brain immune response. This hypothesis predicts that formation of Kupfer-type immunological synapses is necessary for polarized distribution of effector molecules, and their directed secretion toward the target cells. However, no studies have been published testing the hypothesis that cytokines can only form polarized clusters at Kupfer-type immunological synapses. Here, we show that IFN-γ and granzyme-B cluster in a polarized fashion at contacts between T cells and infected astrocytes in vivo. In some cases these clusters were found in Kupfer-type immunological synapses between T cells and infected astrocytes, but we also detected polarized IFN-γ at synaptic immunological contacts which did not form Kupfer-type immunological synaptic junctions, ie, in the absence of polarization of TCR or LFA-1. This indicates that TCR signaling, which leads to the production, polarization, and eventual directed secretion of effector molecules such as IFN-γ, occurs following the formation of both Kupfer-type and non-Kupfer type immunological synaptic junctions between T cells and virally infected target astrocytes in vivo.