We have used a T cell receptor transgenic mouse adoptive transfer system to allow direct visualization of antigen specific T cell activation and cytokine expression in vivo. This system involves the adoptive transfer of 5 x 10' T cells that express the transgenic DO11.10 TCR specific for ovalbumin (OVA) peptide 323-339 to normal syngeneic (BALB/c) mice. Three days after adoptive transfer the mice are immunized with 300 fig OVA peptide in CFA subcutaneously (s.c.). Antigen specific cells are detected with the anti-clonotype mAb KJ1-26. The number of clonotype positive (KJ1-26+) T cells in draining lymph nodes increased eight-fold, reaching a maximum at 72 hours, and returned to baseline over 14 days. More than 90% of KJ1-26+ cells in draining nodes expressed IL-2 receptor from 8-24 hours following immunization. The majority of KJ1-26+ cells progressed from a CD441°/CD45RBhi/L-selectinni phenotype to a CD44hi/CD45Rbl°/L-selectinI° phenotype over 5 days. Expression of mRNA encoding IL-2, IL-4, IL-10, and IFN-y was studied in mice immunized with 300g OVA323-339 in PBS intravenously (i.V.) or in CFA (s.c.) or with 4 mg ovalbumin protein in CFA (s.c.). Following peptide immunization i.V., the percentage of KJ1-26+ cells expressing IL-2 mRNA peaked at 50% at 2 hours and returned to near 0 by 8 hours. Following s.c. injection of peptide in CFA IL-2 expression peaked at 9% at 8 hours and persisted at low levels for 24 hours. IL-2 expressing cells were concentrated at the border between the T cell and B cell regions of the lymph node. Finally, following immunization with ovalbumin in CFA (s.c.), IL-2 expression peaked at 3.5% at 24 hours, but persisted for up to 96 hours. Expression of interferon-y occurred at roughly eight-fold lower frequency and with similar kinetics. No expression of IL-4 or IL-10 was detected following primary immunization.