We have shown that C57BL/6-derived CD8+ CTL specific for an immunodominant herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) determinant express a highly conserved VB1O/junctional sequence combination. This extreme T cell receptor B-chain bias can be used to track the activation of gB-specific CTL in lymph nodes draining the site of HSV-1 infection. In this report we have examined the accumulation of gB-specific CTL in the primary and secondary or recall CTL responses to HSV-1 infection. We found that gB-specific cytolytic activity present within popliteal lymph nodes draining HSV-infected foot-pads peaked at day 5 post-infection during the primary response. As found previously, this correlates with the accumulation of VB1O+CD8+ CTL in the activated T cell subset. Lymph node-derived cytotoxicity peaked between days 3 and 4 on secondary challenge with virus and, somewhat surprisingly, was considerably below that seen in the primary response. This reduced gB-specific cytolytic activity mirrored a near absence of VB1O+CD8+ T cell enrichment found within the draining lymph nodes during this recall response, consistent with the overall diminution of gB-specific CTL accumulation in this site. Finally, there was a second wave of biased accumulation of VB1O+CD8+ activated T cells within the popliteal lymph nodes well after the resolution of infection in both the primary and secondary responses. These results are discussed in terms of preferential activation of virus-specific memory T cells directly in infected tissues during a secondary CTL response at the expense of draining lymphoid organs.