Computer-based database searching and protein multiple sequence alignment has identified a novel clan of zinc metallopeptidases, which, by phylogenetic analysis, has been shown to contain six subfamilies. The family is characterized by four common transmembrane segments and three conserved sequence motifs. The combination of topology analysis and motif identification has detected three potential Zn2+ coordinating residues. Only two of the sequences of this novel zinc metallopeptidase clan possess any functional annotation, one of which is able to cleave its substrate within a cytosol/transmembrane segment junction. A number of observations suggest that the remaining members of this novel clan may also cleave their substrates within transmembrane segments.