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Tail‐anchored (TA) proteins insert into their target organelles by incompletely elucidated posttranslational pathways. Some TA proteins spontaneously insert into protein‐free liposomes, yet target a specific organelle in vivo. Two spontaneously inserting cytochrome b5 forms, b5‐ER and b5‐RR, which differ only in the charge of the C‐terminal region, target the endoplasmic reticulum (ER) or the mitochondrial outer membrane (MOM), respectively. To bridge the gap between the cell‐free and in cellula results, we analyzed targeting in digitonin‐permeabilized adherent HeLa cells. In the absence of cytosol, the MOM was the destination of both b5 forms, whereas in cytosol the C‐terminal negative charge of b5‐ER determined targeting to the ER. Inhibition of the transmembrane recognition complex (TRC) pathway only partially reduced b5 targeting, while strongly affecting the classical TRC substrate synaptobrevin 2 (Syb2). To identify additional pathways, we tested a number of small inhibitors, and found that Eeyarestatin I (ESI) reduced insertion of b5‐ER and of another spontaneously inserting TA protein, while not affecting Syb2. The effect was independent from the known targets of ESI, Sec61 and p97/VCP. Our results demonstrate that the MOM is the preferred destination of spontaneously inserting TA proteins, regardless of their C‐terminal charge, and reveal a novel, substrate‐specific ER‐targeting pathway. [ABSTRACT FROM AUTHOR]