The evolution of the virus-specific cytotoxic T-lymphocyte response in two cats experimentally infected with feline immunodeficiency virus (FIV) was monitored. Effector cells were derived from peripheral blood lymphocytes during the acute and chronic phases of infection (0 to 21 and 62 to 127 weeks, respectively) and from the spleen and lymph nodes at 127 weeks after infection. Lymphocytes were restimulated in vitro with paraformaldehyde-fixed, autologous lymphoblasts which had been infected with recombinant vaccinia viruses expressing FIV GAG or ENV proteins. Unstimulated lymphocytes were also used as effectors in some assays. 51Cr-labelled autologous skin fibroblasts infected with recombinant vaccinia viruses were used as targets. FIV GAG-specific cytotoxic precursors were detected in restimulated circulating lymphocytes during acute infection in both cats. The onset of this activity was as early as 2 weeks postinfection (p.i.) in one cat. From 62 weeks p.i. neither FIV GAG- nor ENV-specific precursors could be detected in the peripheral blood. However, at 127 weeks p.i., GAG- and ENV-specific cytotoxic precursors were detected in lymphocytes isolated from lymph nodes. The FIV-specific cytotoxic cells were predominantly major histocompatibility complex class I restricted. No cytotoxic activity was detected from unstimulated lymphocytes. These studies demonstrate the use of an assay system for dissecting the FIV-specific cytotoxic cell response and show that precursor cells appear in the circulation very early after infection and prior to a detectable antibody response. Our results also suggest that the persistent high-level circulating antiviral cytotoxic T-lymphocyte responses seen in human immunodeficiency virus-infected humans may not be a feature of FIV infections in cats.