This paper presents a way to develop a micropropagation protocol for mature trees of Fraxinus excelsior. Methods for establishment, multiplication, and rooting were optimized by testing various clones, resulting in the successful in vitro propagation of 26 selected mature trees. New cultures were started most successfully from young shoot tips or axillary buds of grafted mature plants. Explants were surface-sterilized by using 0.2% mercury chloride (HgCl₂), prior to the transfer on WPM, without plant growth regulators (PGR), followed by WPM containing 4 mg l^-1 BAP and 0.15 mg l^-1 IBA after two weeks. For some clones the supplement of 0.1 mg l^-1 TDZ to the medium promoted the establishment. The best multiplication rates were obtained on WPM containing 4 mg l^-1 BAP, 0.15 mg l^-1 IBA and 0.01 mg l^-1 TDZ and 0.7% agar in honey jars closed with a plastic lid. As rooting medium half-strength MS medium with 2 mg l^-1 IBA, 0.25 mg l^-1 BAP and 0.8% Difco granulated agar was chosen. In most cases there was no rooting in vitro, but after the transfer to soil and acclimatization the microcuttings developed roots. Mature cultures only rooted using rooting medium with IBA and the cytokinin BAP. Juvenile cultures showed similar rooting with or without the supplement of cytokinin and auxin to the rooting medium..