The mitochondrial inner membrane exhibits a complex topology. Its infolds, the cristae membranes, are contiguous with the inner boundary membrane (IBM), which runs parallel to the outer membrane. Using live cells co-expressing functional fluorescent fusion proteins, we report on the distribution of inner membrane proteins in budding yeast. To this end we introduce the enlarged mitochondria of Delta mdm10, Delta mdm31, Delta mdm32, and Delta mmm1 cells as a versatile model system to study sub-mitochondrial protein localizations. Proteins of the F1F0 ATP synthase and of the respiratory chain complexes III and IV were visualized in the cristae-containing interior of the mitochondria. In contrast, proteins of the TIM23 complex and of the presequence translocase-associated motor were strongly enriched at the IBM. The different protein distributions shown here demonstrate that the cristae membranes and the IBM are functionally distinct sub-compartments. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.