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RNase Sa3 produced by Streptomyces aureofaciens strain CCM 3239 belongs to the T1 family of microbial ribonucleases. It is closely related both to RNase Sa, studied in detail earlier, and to RNase Sa2 produced by the same microorganism. The most important property of RNase Sa3 is the relatively high cytotoxic activity, which was not observed for RNase Sa and Sa2. Recombinant RNase Sa3 was overexpressed in Escherichia coli and purified to high homogeneity. The hanging-drop vapour-diffusion method was used for crystallization. The two crystal forms are trigonal P3121 and tetragonal P41212, with unit-cell parameters a = b = 64.7, c = 69.6 Å, γ = 120° and a = b = 34.0, c = 147.2 Å, respectively. They diffract to 2.0 and to 1.7 Å resolution, respectively, using synchrotron radiation. The asymmetric units of crystal forms I and II contain one molecule of the enzyme, which corresponds to VM = 3.8 Å3 Da−1 …