We have shown previously that it is possible to target complement-mediated killing against cultured ovarian tumour cells in vitro. As malignant ovarian cells usually grow in solid nodules in vivo, we have in the present study examined the effectiveness of complement killing against ovarian teratocarcinoma cells (PA-1) growing in three-dimensional tumour microspheroids (TMSs). Our study shows that PA-1 cells growing in TMSs are less susceptible to complement-mediated killing than cells growing in monolayer cultures, even after neutralization of protectin (CD59), the main inhibitor of complement lysis. Cells in suspension and cells growing in TMSs showed a similar expression of membrane co-factor protein (MCP, CD46) and CD59. Decay-accelerating factor (DAF, CD55) was not detected on the surface of cells in suspension, but appeared focally on the outermost cell layers of the TMSs. Complement-activating antibodies bound to all PA-1 cells in suspension but only to the most peripherally located cells in TMSs, even though the target antigens were similarly expressed in the two systems. Antibody-induced complement activation on PA-1 cells in suspension led to C3 and C5b-9 deposition on most cells, while C3 and C5b-9 were only found on the outermost layers of the TMSs. The increased complement resistance of tumour cells growing in three-dimensional spheroids is partly because of an insufficient penetration of antibodies and complement into the TMSs. TMSs are a useful model for the development of more efficient ways to kill malignant cells in micrometastases with monoclonal antibodies and complement.