α-galactosylceramide (KRN 7000, α-GalCer) has shown potent in vivo anti-tumour activity in mice, including against melanoma and the highly specific effect of inducing proliferation and activation of human Vα24+ NKT-cells. We hypothesized that human Vα24+ NKT-cells activated by α-GalCer might exhibit anti-tumour activity against human melanoma. To investigate this, Vα24+ NKT-cells were generated from the peripheral blood of patients with melanoma after stimulation with α-GalCer pulsed monocyte-derived dendritic cells (Mo-DCs). Vα24+ NKT-cells did not exhibit cytolytic activity against the primary autologous or allogeneic melanoma cell lines tested. However, proliferation of the melanoma cell lines was markedly suppressed by co-culture with activated Vα24+ NKT-cells (mean±SD inhibition of proliferation 63.9±1.3%). Culture supernatants of activated Vα24+ NKT-cell cultures stimulated with α-GalCer pulsed Mo-DCs exhibited similar antiproliferative activities against melanoma cells, indicating that the majority of the inhibitory effects were due to soluble mediators rather than direct cell-to-cell interactions. This effect was predominantly due to release of IFN-γ, and to a lesser extent IL-12. Other cytokines, including IL-4 and IL-10, were released but these cytokines had less antiproliferative effects. These in vitro results show that Vα24+ NKT-cells stimulated by α-GalCer-pulsed Mo-DCs have anti-tumour activities against human melanoma through antiproliferative effects exerted by soluble mediators rather than cytolytic effects as observed against some other tumours. Induction of local cytokine release by activated Vα24+ NKT-cells may contribute to clinical anti-tumour effects of α-GalCer.© 2001 Cancer Research Campaign www. bjcancer. com