The malaria parasite affects millions of people each year, lives and multiplies in two different hosts, and synthesizes a large number of proteases and heat shock proteins (HSPs) for its survival. We describe here the characterization of a metalloprotease activity which resides in the small HSP (PVHSP28) of the common but noncultivable human malaria parasite Plasmodium vivax. The protein is expressed by erythrocytic stages of the parasite. It is expressed as a ~55-kDa polypeptide which is then processed to the 28-kDa mature protein. The latter was found to be an active protease in gelatin zymography. This protease showed its optimal activity at 37°C (pH 7.6). It also retained its proteolytic activity at higher temperatures of up to 55°C. The enzyme belongs to the metalloprotease class, as its proteolytic activity was most effectively blocked by 1,10-phenanthroline and was restored to a maximal level by the addition of zinc metal ions. Inhibitors for the cysteine, serine, and aspartate classes of proteases were ineffective against this enzyme. A homology search indicates that PVHSP28 probably belongs to a new class of HSPs which possess the metalloprotease signature sequence.