The human homologue of the Escherichia coli htrA gene product was identified by the differential display analysis of transcripts expressed in osteoarthritic cartilage. This transcript was identified previously as being repressed in SV40-transformed fibroblasts (Zumbrunn, J., and Trueb, B. (1996) FEBS Lett. 398, 187-192). Levels of HtrA mRNA were elevated approximately 7-fold in cartilage from individuals with osteoarthritis compared with nonarthritic controls. Differential expression of human HtrA protein was confirmed by an immunoblot analysis of cartilage extracts. Human HtrA protein expressed in heterologous systems was secreted and exhibited endoproteolytic activity, including autocatalytic cleavage. Conversion by mutagenesis of the putative active site serine 328 to alanine eliminated the enzymatic activity. Serine 328 was also found to be required for the formation of a stable complex with alpha1-antitrypsin. We have determined that the HtrA gene is highly conserved among mammalian species: the amino acid sequences encoded by HtrA cDNA clones from cow, rabbit, and guinea pig are 98% identical to human. In E. coli, a functional htrA gene product is required for cell survival after heat shock or oxidative stress; its role appears to be the degradation of denatured proteins. We propose that mammalian HtrA, with the addition of a new functionality during evolution, i.e. a mac25 homology domain, plays an important role in cell growth regulation.