The free calcium concentration in nucleus ([Ca2+]n) and in cytoplasm ([Ca2+]c) of single cells were estimated by confocal laser microscopy using the Ca(2+)-indicator Indo-1. It is shown that in various cell types a nucleo-cytosolic Ca(2+)-gradient is present at rest and during stimulation. The direction and the extent of the nucleo-cytosolic Ca(2+)-gradient may vary with the cell type, differentiation status, phosphorylation conditions and also with the type of agonist. Evidence is given for the role of extra- and intranuclear storage sites as well as for Ca(2+)-influx. Finally potential artefactual interference with the measurements is discussed.