Microsomal glutathione transferase 1 (MGST1) is a membrane bound enzyme involved in the detoxification of reactive electrophiles and protection of membranes from oxidative stress. The enzyme displays an unusual and broad subcellular distribution with especially high levels in the endoplasmic reticulum (ER) and outer mitochondrial membrane (OMM). Here we examined the molecular basis for this dual distribution. We hypothesized that the amphipathic properties of the first transmembrane segment (TMS), that contains a positively charged lysine (K25), is a central feature guiding dual targeting. The lysine-25 was substituted to alanine by site directed mutagenesis. We also increased the amphipathic character of the helix by inserting an additional lysine either one turn above or below K25. Expressing these constructs in simian COS cells, and analyzing subcellular distribution by immunocytochemistry, we observed an increased ER targeting of K25A-MGST1. In contrast I22K-MGST1 and F28K-MGST1 displayed pronounced mitochondrial targeting. By using in vitro transcription-translation we examined whether insertion of WT-MGST1 into ER is co- or post-translational and provide evidence for the former. In the same experimental set-up, mitochondrial insertion was shown to depend on the positive charge. Together these results show that removing the positive charge of lysine-25 promotes ER incorporation, but counteracts mitochondrial insertion. In contrast, introducing an extra lysine in the first TMS of MGST1 had opposite effects. The amphipathic character of the first TMS thus constitutes a molecular determinant for the dual targeting of MGST1. Broad subcellular distribution is consistent with a physiological role in protection from reactive intermediates and oxidative stress.