Cell activation resulting from binding of receptors on one cell to ligands on another is governed by receptor affinities and by ligand concentrations. Effective ligand concentration is determined by its density on the cell surface, but receptor occupancy level will also be influenced by the area of surface contact between the cells. The present study demonstrates the critical importance of a large, continuous surface contact area for effective CTL activation. Using class I alloantigen immobilized on latex microspheres, particle sizes of 4 to 5 microns were found to provide an optimum stimulus. Below 4 microns, responses decreased rapidly with decreasing particle size, and large numbers of small particles could not compensate for suboptimal size. Comparable size dependence was found for activation of degranulation by cloned CTL and for stimulation of in vitro generation of CTL responses by spleen cells from in vivo primed mice. In the presence of fluid-phase anti-TCR antibody, CD8-dependent binding to non-Ag class I (i.e., class I that is not recognized by the TCR) can provide a costimulatory signal to activate degranulation. This response is also critically dependent upon the class I being presented on a particle of 4 or 5 microns diameter. The results suggest that sufficient receptor occupancy (both TCR and CD8) over a contiguous region of the cell surface, as opposed to total interactions over the entire cell surface, is a critical determinant for activation. The ability of CTL to distinguish between Ag on cell-size vs subcellular fragments is probably necessary for their effective functioning, and may also explain the inability to significantly influence CTL activation in vivo with subcellular or soluble forms of Ag.