We reported recently that mevalonate kinase (EC 2.7.1.36; ATP:mevalonate 5-phosphotransferase) that was isolated from rat liver and believed to be a cytosolic protein was localized in rat liver peroxisomes. In addition, we found that the mevalonate kinase monoclonal antibody used in the study also reacted with several other proteins present in the mitochondrial and cytosolic fractions. These findings raised the prospect of the presence of several isoenzymes of mevalonate kinase localized in different compartments of the cell. In the current study we produced four new polyclonal antibodies against different epitopes of mevalonate kinase to investigate the subcellular localization of the protein by several different approaches: (i) by analytical subcellular fractionation and immunoblotting of mevalonate kinase in the isolated subcellular fractions with the monospecific antibodies; (ii) by immunocryoelectron microscopy techniques; and (iii) by expressing the cDNA encoding mevalonate kinase in mammalian cells. The data obtained demonstrate that there is only one mevalonate kinase protein that is predominantly localized in peroxisomes. We also illustrate that the protein is targeted to and imported into peroxisomes. In addition, we show that in cells and tissues obtained from patients with peroxisomal deficiency diseases mevalonate kinase protein and its activity are severely reduced.