An experimental setup for measurement of time-resolved autofluorescence of the human eye fundus is demonstrated. The method combines laser scanning technique and time-correlated single photon counting. The light source is a laser diode, delivering pulses of about 100 ps duration at a repetition rate of 40 MHz. The excitation wavelength is 446 nm and the cutoff wavelength of fluorescence detection is at 475 nm. The autofluorescence can be determined with a spatial resolution of 80×80 μm2 and 25 ps time resolution. The fluorescence decay is optimally approximated by a biexponential model. The dominating lifetime τ1 is shortest in the macula (320 to 380 ps) and reaches 1500 ps in the optic disk. The lifetime τ2 varies between 2 ns and 5 ns, but the spatial distribution is more homogeneous. Respiration of 100% oxygen for 6 min leads to changes in the fluorescence lifetime pointing to detection of coenzymes. Diagrams of lifetime τ2 versus τ1 are well suited for comparison of substances. Such lifetime clusters of a 20 deg macular field of a young healthy subject and of a patient suffering from dry age-related macular degeneration overlap only partially with τ2-τ1 clusters of lipofuscin. © 2004 Society of Photo-Optical Instrumentation Engineers.