In this report the V-H and V-H genes of the anti-T cell receptor (TCR) antibody KJ16, which recognizes the TCR V beta 8.1 and V beta 8.2 regions in mice, were cloned and expressed as a single-chain antibody (scFv) in Escherichia coli. A 29-kDa protein was obtained after renaturation from inclusion bodies. The KJ16 scFv bad a relative affinity for the native TCR that was slightly higher than KJ16 Fab fragments. The scFv and Fab fragments of the KJ16 antibody, together with monovalent forms of two other anti-TCR antibodies, were evaluated as antagonists of the T cell-mediated recognition of a peptide-class I complex or of a superantigen. Staphylococcus enterotoxin B (SEB) bound to a class II product. Each of the anti-TCR antibodies was efficient at inhibiting the recognition of the SEE-class II complex. In contrast, only the clonotypic antibody, which binds to epitopes on both the V beta and V alpha regions, inhibited the recognition of peptide-class I complex. We conclude that the TCR binding site for the SEB-class II ligand encompasses a larger surface area than the TCR binding site for the peptide-class I ligand.