Copyright © by American Society for Cell Biology.-- Final full-text version of the paper available at: http://www.molbiolcell.org/content/vol15/issue3/3-Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is a key enzyme in the sterol biosynthesis pathway, but its subcellular distribution in the Trypanosomatidae family is somewhat controversial. Trypanosoma cruzi and Leishmania HMGRs are closely related in their catalytic domains to bacterial and eukaryotic enzymes described but lack an amino-terminal domain responsible for the attachment to the endoplasmic reticulum. In the present study, digitonin-titration experiments together with immunoelectron microscopy were used to establish the intracellular localization of HMGR in these pathogens. Results obtained with wild-type cells and transfectants overexpressing the enzyme established that HMGR in both T. cruzi and Leishmania major is localized primarily in the mitochondrion and that elimination of the mitochondrial targeting sequence in Leishmania leads to protein accumulation in the cytosolic compartment. Furthermore, T. cruzi HMGR is efficiently targeted to the mitochondrion in yeast cells. Thus, when the gene encoding T. cruzi HMGR was expressed in a hmg1 hmg2 mutant of Saccharomyces cerevisiae, the mevalonate auxotrophy of mutant cells was relieved, and immunoelectron analysis showed that the parasite enzyme exhibits a mitochondrial localization, suggesting a conservation between the targeting signals of both organisms.This work was supported by grants from the UNDP/World Bank/World Health Organization Program for Research and Training in Tropical diseases (T24/181/30 ID 980139), the Spanish Programa Nacional de Biotecnología (BIO97-0659), the EC INCO-DC project contract no. CT980371, and the Plan Andaluz de Investigación (Cod. CVI-199). J.P. and R.H. are fellows of the Spanish PFPI of the Ministerio de Educación y Ciencia, A.M. and C.F.L. had a fellowship from the Instituto deCooperacion Iberoamericana (Spain).Peer reviewed