Ancestral sequence reconstruction is a powerful approach allowing the resurrection of ancient enzymes based on sequences predicted by a phylogenetic analysis [1]. This paleomolecular approach uncovers the properties of the ancestral proteins, indicating the structural consequences of the molecular evolution [2]. In this project the sequences of representative members of haloalkane dehalogenase subfamily II were selected as targets for prediction of common ancestor of haloalkane dehalogenase DbjA [3–5] and DbeA [6], ancDbjA-DbeA node1, and additional ancestors corresponding to the deeper nodes of the branch leading towards the present-day enzymes, ancDbjA-DbeA node2, ancDbjA-DbeA node3, ancDbjA-DbeA node4 and ancDbjA-DbeA node5. The genes encoding predicted sequences were synthesized; the resurrected proteins were overexpressed in Escherichia coli BL21(DE3) cells and purified to homogeneity by metallo-affinity chromatography. All crystallization trials were performed by using the sitting-drop vapor-diffusion method at 23 °C. The crystals of ancDbjA-DbeA node 2, node 3 and node 5 grew during the initial screening and no further optimization of the crystallization conditions was necessary. The triangular prism shaped crystals of ancDbjA-DbeA node3 with dimensions 0.5 x 0.09 x 0.08 mm grew in condition No. 42 of JCSG consisting of 0.02 M magnesium chloride, 0.1 M Tris pH 8.5 and 20% (w/v) PEG 8000. The hexagonally shaped crystals of ancDbjA-DbeA node3 with dimensions 0.2 x 0.1 x 0.04 mm appeared in condition No. 16 of the Wizard classic consisting of 100 mM potassium phosphate/sodium phosphate pH 6.2 and 2.5 M sodium chloride. The trigonal shaped crystals of ancDbjA-DbeA node5 with average dimension 0.11 x 0.05 x 0.31 mm were observed in condition No. 73 of PEG suite containing 0.2 M magnesium acetate and 20 % (w/v) PEG 3350. These crystals were used for collection of X-ray diffraction data and complete diffraction data sets were collected at 1.66, 1.26 and 1.25 Å resolution for ancDbjA-DbeA node2, ancDbjA-DbeA node3 and ancDbjA-DbeA node 5, respectively. Obtained microcrystals of ancDbjA-DbeA node1 and ancDbjA-DbeA node4 were further optimized by variation of enzyme concentration, pH and precipitant concentration. Optimized crystals of ancDbjA-DbeA node4 appeared within three days from the drop composed of 9.5 % (w/v) mix of PEG 1000, PEG 3350 and MPD, 0.1 M MOPS/HEPES-Na pH 7.0. On-going structural analysis of ancestral enzymes will provide insight into their unique catalytic properties such as high thermodynamic stability and high catalytic activity.