HLA/peptide tetramers are frequently used for ex vivo monitoring of disease- or vaccine-induced T cell immune responses and for T cell epitope identification. However, when low-levels HLA/peptide tetramer-positive T cell populations are encountered, it is difficult to ascertain whether this represents a true T cell receptor (TCR)-mediated interaction or background signal. To address this issue, we have developed a method for both HLA class I and class II tetramer assays to confirm tetramer-binding to the TCR/CD3 complex. Preincubation of T cells with anti-CD3 mAb SPV-T3b and subsequent crosslinking interferes with the binding of HLA/peptide tetramers to the TCR/CD3 complex and thereby indicates to what extent HLA/peptide tetramer binds through interaction with TCR/CD3 complex. SPV-T3b pretreatment results in a 2- to 10-fold decrease in tetramer-binding intensity to antigen-specific CD8+ or CD4+ T cells, whereas background reactivity of HLA/peptide tetramers containing HIV-derived peptide in HIV-negative donors remained unchanged. SPV-T3b pretreatment forms a valuable tool to verify tetramer-based detection of antigen-specific T cells during the monitoring of immune responses in clinical studies.