Bothroalternin (MW 27 kDa), a new member of the family of C-type lectins is a thrombin inhibitor which was purified from pooled B. alternatus venom by affinity chromatography on PPACK-thrombin-Sepharose, followed by size exclusion and reverse-phase on HPLC columns. Material retained on the affinity column contained proteins with apparent molecular weights ranging from 20 to 60 kDa on SDS-PAGE and inhibited aggregation of rabbit platelets induced by alpha-thrombin (IC50 = 28 microg/ml). A single band of approximately 27 kDa was recognized in Western-blot assays using polyclonal antibodies raised against bothrojaracin, a thrombin inhibitor purified from B. jararaca venom (Zingali et al., 1993). The immunological similarity of this fraction to bothrojaracin was confirmed by ELISA and competitive ELISA. Further purification by size exclusion and reverse-phase on HPLC, produced a single homogenous peak called bothroalternin. This protein was highly homologous to bothrojaracin (95% in its N-terminal sequence-for residues 1 to 25) but displaying lower inhibitory effect on thrombin induced platelet aggregation (Ic50 = 0.19 microg/ml) compared to bothrojaracin (IC50 = 0.06). Altogether, bothroalternin is a new thrombin inhibitor isolated from Bothrops alternatus venom and has been characterized as a bothrojaracin-like protein.