During investigations into the behaviour and fate of ovalbumin (OVA)-specific CD8+ T cells in a TCR transgenic system, cytotoxic T lymphocyte (CTL) activity (as measured by classical in vitro stimulation followed by 51Cr-release assay) was found to be reduced after OVA peptide administration. This paradoxically occurred even during the peak of activation and expansion of these T cells. The reduced responsiveness occurred for both classical CTL assays and in vitro proliferation assays, and would apparently be consistent with induction of anergic or suppressor T cells. Instead, we provide evidence that in vivo peptide treatment generated activated killers which consequently killed the stimulator cells during in vitro culture, thus resulting in the unresponsive phenotype. In co-culture experiments, the proliferation and classical CTL activity were severely reduced when naive OVA-specific CD8+ T cells (OT-I) cells were co-cultured with cells from OVA peptide-treated mice. Moreover, cells obtained after peptide injection did not require in vitro stimulation to be able to kill target cells. Therefore, activation of killers in vivo should be considered as one pathway whereby unresponsiveness is found in assays requiring in vitro stimulation.