Tim44 is an essential component of the translocase of the inner mitochondrial membrane (TIM) complex that mediates transport of nuclear encoded mitochondrial precursors across the inner membrane. Here. we have investigated the topology of Tim44 by probing mitochondria with membrane impermeable 3-(N-maleimidopropionyl)biocytin (MPB) followed by the specific immunoprecipitation of modified proteins. Our data indicate that a single cysteine residue. Cys-369. located in the C-terminal domain of the yeast Tim44 is exposed to the mitochondrial intermembrane space. (C) 2002 Elsevier Science (USA). All rights reserved.