Beyond antigen processing and presentation, antigen-presenting cells must convey additional signals that are necessary for T-cell activation and proliferation. In this study we analyzed the accessory function of human Langerhans cells (LC) in the primary T-cell response to alloantigens. We used paraformaldehyde-fixed LC suspensions to dissociate membrane-associated accessory structures from soluble activating factors. When freshly prepared LC-enriched (eLC, 8-20% LC) epidermal cell suspensions were fixed, normal allogeneic T cells failed to proliferate. This defect cannot be overcome by the addition of either interleukin (IL)-1 and/or IL-6 during the mixed skin cell lymphocyte reaction (MSLR). By contrast, when eLC were first incubated for 3 d, and especially in the presence of interferon gamma (IFN-gamma) before fixation, they retained significant allostimulatory property that did not correlate with increased MHC class II antigen expression. Furthermore, we showed that once LC have been activated, IL-1, but not IL-6, could serve as a co-stimulatory factor in the primary allogeneic T-cell response. In conclusion, the data suggest that human LC accessory function is not constitutive but requires an activation step, which can be provided by IFN-gamma during LC-T-cell interaction.