An apparatus which allows precise control of the time of initiation and the area of contact of cells with immobilized ligands has been developed. Cells are trapped in an asymmetric film that can be quantitatively thinned, forcing the cells into close contact with ligands adsorbed on the base of the apparatus. Using microbeads to indicate the film height, the amount of thinning can be controlled to within 1 microm, producing known contact areas between cells and the ligand-coated surface. This system was used with anti-CD3-coated surfaces of different densities to examine the effect of ligand density on T cell activation, while keeping the number of ligands presented to the cells constant. T cell activation was observed individually in each cell as intracellular calcium mobilization. In these experiments both the percent of T cell activation and the rate of calcium rise were found to depend only on the number of anti-CD3 molecules presented and not on their density. This implies that the spacing between molecules is not important in the range studied, and suggests that receptor clustering to levels higher than dimers may not be necessary for induction of calcium mobilization by anti-CD3.