Fresh fruiting bodies of the mushroom Calvatia caelata, commonly known as the mosaic puffball, were extracted with 10 mM Tris-HCl buffer (pH 7.2). The extract was subjected to a chromatographic procedure which has been successfully used to isolate ribosome-inactivating proteins from plants. It was first applied to an Affi-gel blue gel column previously equilibrated and eluted with 10 mM Tris-HCl buffer. The adsorbed fraction eluted with 1.5 M NaCl in the buffer was then purified by chromatography on DEAE-cellulose in the same buffer. The unadsorbed fraction yielded essentially a single peak when it was chromatographed on a column of Mono S. When the peak was then gel filtered on Superdex 75, it yielded a homogeneous peak with a molecular mass of 39 kDa. The protein, which was designated calcaelin, was dissociated into two subunits with a molecular mass of 19 kDa and 20 kDa, respectively, in SDS-polyacrylamide gel electrophoresis. The two bands exhibited the same N-terminal amino acid sequence which was somewhat similar to those of plant ribosome-inactivating proteins. A lesser degree of sequence homology to the fungal ribosome inactivating proteins alpha-sarcin and restrictocin was detected. Calcaelin inhibited translation in rabbit reticulocyte lysate with an IC 50 value of 4 nM and displayed a heat-labile RNase activity of 1.58 U/mg toward yeast tRNA. It exhibited an antimitogenic activity toward mouse splenocytes, and it reduced the viability of breast cancer cells. There was no hemagglutinating, antibacterial or antifungal activity.