In the past decade several reports have claimed that peroxisomes play a critical role in the isoprenoid/cholesterol biosynthetic pathway based on the finding of a predominant peroxisomal localization of several of the enzymes involved. Other reports, however, do not support the peroxisomal localization of these enzymes. In this study we have studied the subcellular localization of one of the enzymes, human mevalonate pyrophosphate decarboxylase, by conventional subcellular fractionation and digitonin permeabilization studies, immunofluorescence microscopy, and immunoelectron microscopy. We found a cytosolic localization for both endogenous human mevalonate pyrophosphate decarboxylase (in human fibroblasts, liver, CV1 and HEK293 cells) and overexpressed mevalonate pyrophosphate decarboxylase (in human fibroblasts, HEK293 and CV1 cells) but no indication for a peroxisomal localization. Our results do not support a central role of peroxisomes in the isoprenoid/cholesterol biosynthetic pathway.