Calcium (Ca(2+)) is a ubiquitous intracellular second messenger and involved in a plethora of cellular processes. Thus, quantification of the intracellular Ca(2+) concentration ([Ca(2+)]i) and of its dynamics is required for a comprehensive understanding of physiological processes and potential dysfunctions. A powerful approach for studying [Ca(2+)]i is the use of fluorescent Ca(2+) indicators. In addition to the fluorescence intensity as a common recording parameter, the fluorescence lifetime imaging microscopy (FLIM) technique provides access to the fluorescence decay time of the indicator dye. The nanosecond lifetime is mostly independent of variations in dye concentration, allowing more reliable quantification of ion concentrations in biological preparations. In this study, the feasibility of the fluorescent Ca(2+) indicator Oregon Green Bapta-1 (OGB-1) for two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was evaluated. In aqueous solution, OGB-1 displayed a Ca(2+)-dependent biexponential fluorescence decay behaviour, indicating the presence of a Ca(2+)-free and Ca(2+)-bound dye form. After sufficient dye loading into living cells, an in situ calibration procedure has also unravelled the Ca(2+)-free and Ca(2+)-bound dye forms from a global biexponential fluorescence decay analysis, although the dye's Ca(2+) sensitivity is reduced. Nevertheless, quantitative [Ca(2+)]i recordings and its stimulus-induced changes in salivary gland cells could be performed successfully. These results suggest that OGB-1 is suitable for 2P-FLIM measurements, which can gain access to cellular physiology.