Cancer immunotherapy aims at inducing immune responses against tumour-associated antigens that mediate the eradication of tumour cells. For successful vaccination against antigens expressed by the tumour, the immune system has to be provided with sufficient amounts of these antigens in connection with strong immunostimulatory signals such as toll-like receptor (TLR) ligands. Tumour cells represent a convenient source of relevant tumour-associated antigens but can have suppressive properties. In this study, we explored how different forms of tumour cell material influence the activation of dendritic cells (DC), which play a crucial role in the induction of anti-tumour immune responses. We show that freeze-and-thaw-disrupted tumour cells inhibit DC activation in response to TLR stimulation, a phenomenon that is only partially seen with non-disrupted control cells. This suppression of DC stimulation is independent of tumour cell- and species-specific factors. We tested the hypothesis that phosphatidylserine on cells with disrupted membrane integrity mediates inhibition of TLR-induced DC activation. Our experimental evidence indicates that phosphatidylserine is not involved in the inhibition of TLR-mediated DC activation by freeze-and-thaw-disrupted cells. The inhibitory activity associated with disrupted tumour cells could explain why such preparations are less effective tumour vaccines than apoptotic tumour cells.