A new zinc metalloendopeptidase that cleaves peptides on the amino-terminal side of aspartic acid was isolated from the cultural filtrate of Flavobacterium meningosepticum. The gene for this new enzyme was cloned into pBluescript, and the complete nucleotide sequence was determined. Over 40% of the deduced amino acid sequence was verified independently by direct protein microsequencing. The most important structural features of this new enzyme include (i) the presence of an unusual O-linked oligosaccharide of unknown function located at a unique consensus site near the C-terminus and (ii) a characteristic extended zinc-binding site and corresponding Met-turn that places this metalloendopeptidase in the astacin family. This is the first example of a prokaryotic enzyme related to the eukaryotic astacin group; it is being designated hereafter as flavastacin.