To fill the unmet need for improved matrixes for matrix-assisted laser desorption ionization (MALDI) tissue imaging of small molecules, dithranol (DT)-a matrix mainly used for the analysis of synthetic polymers-was evaluated for detection of lipids in rat liver and bovine calf lens, using MALDI Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). The use of DT resulted in better detection of endogenous lipids than did two other commonly used matrixes, α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), with >70 lipid entities (including phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, phosphatidylserines, phosphatidylglycerol, phosphatidic acids, ceramide phosphates, sterol lipids, acyl carnitines, and glycerides) being detected in rat liver and bovine lens tissue sections, using positive-ion detection. Using saturated DT in chloroform/methanol (2:1, v/v), with 1% formic acid in the final mixture, 57 lipid entities were successfully imaged from bovine calf lens, with clear and distinct distribution patterns. In a section across the lens equatorial plane, all compounds showed concentric distributions around the lens nucleus and most showed specific abundance changes, which correlated with lens fiber cell age. As a novel finding, palmitoylcarnitine and oleoylcarnitine were found uniquely localized to the younger lens fiber cell cortex region. This work demonstrates the potential of DT as a new matrix for tissue imaging by MALDI-FTICR MS.