A fast fluorescence lifetime imaging (FLIM) system is developed that can acquire images at a rate of hundreds of frames per second. The FLIM system is based on a wide-field microscope equipped with a time-gated intensified CCD detector and a pulsed laser. The time-gated detector acquires the signals from two time gates simultaneously and is therefore insensitive to movements of the specimen and photo-bleaching. The system is well suited for quantitative biological FLIM experiments and its performance is evaluated in calcium imaging experiments on beating neonatal rat myocytes. Several calcium sensitive dyes are characterized and tested for their suitability for fast FLIM experiments: Oregon Green Bapta-1 (OGB1), Oregon Green Bapta-2 (OGB2), and Oregon Green Bapta-5N (OGB5N). Overall the sensitivity range of these dyes is shifted to low calcium concentrations when used as lifetime dyes. OGB1 and OGB2 behave very similarly and can be used for FLIM-based calcium imaging in the range 1 to ∼500 nM and OGB5N can be used up to 3 μM. The fast FLIM experiments on the myocytes could be carried out at a 100-Hz frame rate. During the beating of the myocytes a lifetime change of about 20% is observed. From the lifetime images a rest calcium level of about 65 nM is found. © 2004 Society of Photo-Optical Instrumentation Engineers.