A lithography tool is calibrated using a calibration substrate having a set of first marks distributed across its surface in a known pattern. The tool is operated to apply a pattern comprising a plurality of second marks at various positions on the substrate, each second mark overlying one of the first marks and being subject to an overlay error dependent on an apparatus-specific deviation. The second marks are applied by multiple exposures while the substrate remains loaded in the tool. An operating parameter of the apparatus is varied between the exposures. An overlay error is measured and used to calculate parameter-specific, apparatus-specific calibration data based on knowledge of the parameter variation used for each exposure.