We have analyzed the role of the highly abundant molecular chaperone Hsp60 in the biogenesis of medium-chain acyl-CoA dehydrogenase (MCAD) using RNA interference (RNAi). MCAD is a mitochondrial enzyme involved in the fatty acid metabolism and previous studies in isolated rat mitochondria or prokaryotic expression systems have shown that Hsp60 and GroEL are involved in the folding of MCAD proteins. To elucidate the impact of Hsp60 levels for folding and assembly of MCAD proteins in intact mammalian cells, we report the design and in vivo synthesis of anti-human Hsp60 small-hairpin RNAs (shRNAs). Quantitative PCR analysis of transfected HEK-293 cells showed significant down-regulation of endogenous Hsp60 mRNA 48 h post-transfection and Western blot analysis confirmed the reduced levels of Hsp60 protein. Furthermore, expression of exogenous Myc-tagged Hsp60 was decreased in shRNA-transfected cells. Flow cytometry showed that shRNA-treatment only affects green fluorescent protein targeted to mitochondria, demonstrating that the shRNA effect is specific. In cells with reduced Hsp60 levels both the amounts of total MCAD proteins and folded MCAD were reduced for MCAD wild-type and the two disease-associated variants studied. A similar effect was observed in cells expressing mitochondrial short-chain acyl-CoA dehydrogenase. Thus, in intact human cells we demonstrate that Hsp60 is involved in the folding of MCAD variant proteins. The present system can be used to study the requirement of Hsp60 for folding of other mitochondrial proteins and to assess the role of Hsp60 for the severity of genetic defects involving these proteins.