CD44 is a multifunctional adhesion molecule that has been shown to be a costimulatory factor for T-cell activation in vitro and in vivo. The aim of the present study was to expand these findings by characterizing the role of CD44 during dendritic cell (DC) antigen presentation to naive, resting T cells. Certain monoclonal antibodies (mAbs) directed against all CD44 isoforms (pan CD44), or against the epitope encoded by the alternatively spliced exon v4 (CD44v4), dose-dependently inhibited the capacity of murine DC to induce proliferation of naive alloreactive T cells. Preincubation of the T cells or DC with these CD44 mAbs revealed that the effect was dependent upon mAb binding to DC, but not to T cells. DC treated with anti-pan CD44 and anti-CD44v4 mAbs induced CD4+ T-cell apoptosis, as shown by annexin V staining and TdT-mediated biotin-dUTP nick-end labelling (TUNEL) assays. However, CD4+ T-cell apoptosis was not dependent on the Fas/Fas ligand (Fas/FasL) system, as DC from FasL-deficient (Gld) mice and T cells from Fas-deficient (Lpr) mice were still susceptible to apoptosis induced by CD44-treated DC. To investigate whether CD44 treatment of DC affects early T-cell/DC interactions, time-lapse video microscopy was performed using peptide-specific T cells from T-cell receptor (TCR) transgenic mice. Interestingly, calcium signalling in CD4+ T cells was significantly diminished following interaction with CD44 mAb-treated DC, but this was not observed in CD8+ T cells. Taken together, we found that perturbation of distinct epitopes of CD44 on DC interfere with early Ca2+ signalling events during the activation of CD4+ T cells, resulting in T-cell apoptosis.