AbstractWe describe a quantitative fluorescence projection tomography technique which measures the 3‐D fluorescence lifetime distribution in optically cleared specimens up 1 cm in diameter. This is achieved by acquiring a series of wide‐field time‐gated images at different relative time delays with respect to a train of excitation pulses, at a number of projection angles. For each time delay, the 3‐D time‐gated intensity distribution is reconstructed using a filtered back projection algorithm and the fluorescence lifetime subsequently determined for each reconstructed horizontal plane by iterative fitting to a mono‐exponential decay. Due to its inherently ratiometric nature, fluorescence lifetime is robust against intensity based artefacts as well as producing a quantitative measure of the fluorescence signal. We present a 3‐D fluorescence lifetime reconstruction of a mouse embryo labelled with an alexa‐488 conjugated antibody targeted to the neurofilament, which clearly differentiates between the extrinsic label and the autofluorescence, particularly from the heart and dorsal aorta. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)