Metalloproteases comprise a diverse superfamily of proteases with a requirement for a metal ion in their catalytic site. These proteins have been shown to play a role in many biological processes including fertilization, cellular adhesion, development, neurogenesis, and metastasis. To help elucidate the molecular mechanisms of metalloprotease function, a strategy was developed to detect and characterize metalloprotease activity in protein extracts prepared from the nematode Caenorhabditis elegans. Extensive biological characterization of C. elegans as well as the availability of the complete genome sequence makes this an excellent model system to study the functions of gene products. Metalloprotease activity was detected in protein extracts prepared from C. elegans assayed in the presence of the fluorescent metalloprotease substrate 2-aminobenzoyl-Ala-Gly-Leu-Ala-4 nitrobenzylamide (AAGLAN). The specificity of this metalloprotease activity was characterized using the inhibitory compounds Phenantrolin (30 mM), a metal chelator, and Phosphoramidon (100 μM), a specific inhibitor, which reduced metalloprotease activity in these extracts 96% and 59% respectively. Characterization of this metalloprotease activity may help to elucidate the role of these proteins in fundamental biological processes and abnormal disease conditions in C. elegans. Multiple sequence alignments of the C. elegans astacin, matrixin, and reprolysin metalloproteases revealed highly conserved metal binding and catalytic domains. Phylogenetic trees of these nematode metalloproteases revealed multiple major subfamilies, whose members likely contribute to the enzyme activity detected. These experiments provide a link to couple biochemical and pharmacological characterization with the genetics and biology of metalloproteases in C. elegans and may help to elucidate their biological functions.