Efferent lymphocytes collected from a cannulated lymphatic draining single lymph nodes were studied for their cytotoxic activity following the injection of live vaccinia virus subcutaneously into the drainage site of a lymph node. Injection of virus produced a 40-fold increase in the lymphoblast output 7 days following virus injection. Cytotoxic lymphocytes were detectable in lymph shortly after the appearance of lymphoblasts at 80 h and also reached a maximum during the 7th day. This was followed by a rapid decline of the cytotoxic cells although cytotoxic cells were detectable up to 2 weeks. The cytotoxic activity in lymph was found to be antigen specific, dependent on the effector/target cell ratio, and allogeneically restricted, indicating that it was most likely due to cytotoxic T lymphocytes (CTL). CTL precursors were found in large numbers in efferent lymph and appeared at approximately the same time as the mature CTL. Unlike CTL, the precursors became part of the recirculating lymphocyte pool and were detectable in efferent lymph for at least 2 months. Following a secondary challenge with vaccinia, lymphoblasts and CTL appeared at least 36 h earlier in the lymph. In summary, we have demonstrated that virus specific CTL are found in the efferent lymph collected from a single immunized lymph node in sheep. The kinetics of the CTL and CTL precursors indicate that these lymphocytes are one of the earliest antigen specific cells detectable in efferent lymph and suggests that these cells migrate rapidly from the lymph node into efferent lymph for dissemination throughout the host to sites of virus infection.