The subcellular location of two integral membrane-bound fatty acid desaturases (Fads), Fad2 and Fad3, was elucidated by immunofluorescence microscopic analyses of tobacco suspension cells transiently transformed with different epitope-tagged versions of the enzymes. Both myc- or hemagglutinin-tagged Fad2 and Fad3 localized to the same region of the endoplasmic reticulum (ER), as evidenced by their co-localization with the ER lumenal protein calreticulin. Results from differential permeabilization experiments revealed that the N-termini of both epitope-tagged Fad2 and Fad3 were exposed on the cytosolic side of ER membranes. These data define the subcellular location and topological orientation of plant desaturases in ER membranes.