Abstract Bacterial hsp60 proteins are major targets of immune responses during infection, and the highly conserved nature of bacterial and mammalian hsp60 has led to speculation that immune reactivity to these stress proteins may be a component of certain autoimmune diseases. We have developed recombinant proteins and monoclonal antibodies to facilitate furthur study of human hsp60 and its association with disease. The human hsp60 gene was expressed in Escherichia coli and a method for purification of the recombinant protein essentially devoid of E. coli GroEL was developed. Using the purified protein we have generated a number of monoclonal antibodies which are specific for human hsp60 and do not cross-react with its counterparts from E. coli and mycobacteria. A highly sensitive sandwich ELISA was developed to quantitate hsp60 levels and was used to study hsp60 accumulation in cells due to vaccinia virus infection and heat shock. This ELISA will be useful for monitoring hsp60 levels in body fluids or tissues during autoimmune reactions and/or inflammatory responses.