Adsorption characteristics of calcium ions on silicon nitride surfaces have been investigated using microcantilevers. Both resonance frequency and cantilever bending undergo variation due to calcium ion adsorption. The adsorption behavior was found to fit a Langmuir isotherm. From the linearized form of the isotherm, the binding constant of adsorption, K, was found to be 116 M-1 and the stress change at maximum coverage, Deltasigma(max), was 0.46 N m(-1). The Gibbs free energy change was estimated to be -11.8 kJ mol(-1). A calcium-binding protein, calmodulin, was used to check for the presence of adsorbed Ca2+ ions on the cantilever surface. Fluorescence measurements with Alexa Fluor 488 conjugated calmodulin gave confirmatory evidence of the presence of Ca2+ ions on the silicon nitride side of the cantilever surface. These results indicate that the presence, of Ca2+ ions and probably other cations in the solution can cause cantilever resonance frequency shifts and bending. It is therefore critical to correct for these contributions even when the reaction of interest is confined to the gold side.