Tail-anchored (TA) proteins are bound to membranes by a hydrophobic sequence located very close to the C-terminus, followed by a short luminal polar region. Their active domains are exposed to the cytosol. TA proteins are synthesized on free cytosolic ribosomes and are found on the surface of every subcellular compartment, where they play various roles. The basic mechanisms of sorting and targeting of TA proteins to the correct membrane are poorly characterized. In mammalian cells, the net charge of the luminal region determines the sorting to the correct target membrane, a positive charge leading to mitochondria and negative or null charge to the endoplasmic reticulum (ER). Here sorting signals of TA proteins were studied in plant cells and compared with those of mammalian proteins, using in vitro translation-translocation and in vivo expression in tobacco protoplasts or leaves. It is shown that rabbit cytochrome b5 (cyt b5) with a negative charge is faithfully sorted to the plant ER, whereas a change to a positive charge leads to chloroplast targeting (instead of to mitochondria as observed in mammalian cells). The subcellular location of two cyt b5 isoforms from Arabidopsis thaliana (At1g26340 and At5g48810, both with positive net charge) was then determined. At5g48810 is targeted to the ER, and At1g26340 to the chloroplast envelope. The results show that the plant ER, unlike the mammalian ER, can accommodate cytochromes with opposite C-terminal net charge, and plant cells have a specific and as yet uncharacterized mechanism to sort TA proteins with the same positive C-terminal charge to different membranes.