Calcium (Ca2+) is s a key component of the signalling network by which plant cells respond to developmental and environmental signals. A change in guard cell cytosolic free Ca2+ ([Ca2+]cyt) is an early event in the response of stomata to both opening and closing stimuli and cyclic nucleotide-mediated Ca2+ signalling has been implicated in the regulation of stomatal aperture. A range of techniques have been used to measure [Ca2+]cyt in plant cells. Here we describe a describe a potential method for imaging cyclic nucleotide-induced changes in [Ca2+]cyt in guard cells using the cameleon ratiometric Ca2+ reporter protein.