By means of Superdex 75 gel filtration and Mono Q ion exchange chromatography, we have isolated a novel platelet aggregation inducer, termed rhodoaggregin, from the crude venom of Calloselasma rhodostoma (Malayan pit viper). The native molecular mass of rhodoaggregin (as estimated by gel filtration chromatography) was 66 kDa while its isoelectric point (pI) was determined to be 3.45. Under reducing conditions of SDS-PAGE, rhodoaggregin exhibited two distinctive bands with molecular masses of 18 kDa (α subunit) and 15 kDa (β subunit). In the absence of reducing agents, however, two bands were also observed, but with apparent molecular masses of 28 (major) and 52 (minor) kDa, respectively. Furthermore, mass spectrometric analysis also showed that rhodoaggregin had a molecular mass of 30155.39±3.25. These molecular weight data suggest that rhodoaggregin probably exists as a tetrameric structure consisting of two disulfide-linked heterodimers. N-terminal amino acid sequence analysis showed that the two subunits of rhodoaggregin exhibit a high degree of homology with each other and with those of the C-type lectin related proteins (CLPs) from other snake venoms. Functional platelet assays showed that rhodoaggregin induced platelet aggregation in rabbit and human whole blood, platelet-rich-plasma (PRP) and washed platelets with a lag period and in an all-or-none manner. The concentration of rhodoaggregin that induced maximal aggregation is estimated at about 0.04 μg/ml (or 0.7 nM).