This study describes the first isolation of hemorrhagic metalloproteinase HR1b from the venom of Okinawa habu, and its cDNA cloning. The substrate specificity of isolated HR1b definitely differed from that of HR1a, further supporting the presence of a metalloproteinase distinguishable from HR1a in the venom of Okinawa habu. The deduced amino acid sequence of HR1b showed 99.67% identity with HR1b of Amami habu, with only two amino acid residue replacements.