Functional alterations are first signs of a starting pathological process. A device that measures parameter for the characterization of the metabolism at the human eye‐ground would be a helpful tool for early diagnostics in stages when alterations are yet reversible. Measurements of blood flow and of oxygen saturation are necessary but not sufficient. The new technique of auto‐fluorescence lifetime measurement (FLIM) opens in combination with selected excitation and emission ranges the possibility for metabolic mapping. FLIM not only adds an additional discrimination parameter to distinguish different fluorophores but also resolves different quenching states of the same fluorophore. Because of its high sensitivity and high temporal resolution, its capability to resolve multi‐exponential decay functions, and its easy combination with laser scanner ophthalmoscopy, multi‐dimensional time‐correlated single photon counting was used for fundus imaging. An optimized set up for in vivo lifetime measurements at the human eye‐ground will be explained. In this, the fundus fluorescence is excited at 446 or 468 nm and the time‐resolved autofluorescence is detected in two spectral ranges between 510 and 560 nm as well as between 560 and 700 nm simultaneously. Exciting the fundus at 446 nm, several fluorescence maxima of lifetime t₁ were detected between 100 and 220 ps in lifetime histograms of 40° fundus images. In contrast, excitation at 468 nm results in a single maximum of lifetime t₁ = 190 ± 16 ps. Several fundus layers contribute to the fluorescence intensity in the short‐wave emission range 510–560 nm. In contrast, the fluorescence intensity in the long‐wave emission range between 560 and 700 nm is dominated by the fluorescence of lipofuscin in the retinal pigment epithelium. Comparing the lateral distribution of parameters of a tri‐exponential model function in lifetime images of the fundus with the layered anatomical fundus structure, the shortest component (t₁ = 190 ps) originates from the retinal pigment epithelium and the second lifetime (t₂ = 1,000 ps) from the neural retina. The lifetime t₃ ≈ 5.5 ns might be influenced by the long decay of the fluorescence in the crystalline lens. In vitro analysis of the spectral properties of expected fluorophores under the condition of the living eye lightens the interpretation of in vivo measurements. Taking into account the transmission of the ocular media, the excitation of NADH is unlikely at the fundus. Microsc. Res. Tech., 2007. © 2007 Wiley‐Liss, Inc.