TetA specified by Tn10 is a class B member of a group of related bacterial transport proteins of 12 transmembrane alpha helices that mediate resistance to the antibiotic tetracycline. A tetracycline-divalent metal cation complex is expelled from the cell in exchange for a entering proton. The site(s) where tetracycline binds to this export pump is not known. We found that, when chelated to tetracycline, Fe(2+) cleaved the backbone of TetA predominantly at a single position, glutamine 225 in transmembrane helix 7. The related class D TetA protein from plasmid RA1 was cut at exactly the same position. There was no cleavage with glycylcycline, an analog of tetracycline that does not bind to TetA. The Fe(2+)-tetracycline complex was not detectably transported by TetA. However, cleavage products of the same size as with Fe(2+) occurred with Co(2+), known to be cotransported with tetracycline. The known substrate Mg (2+)-tetracycline interfered with cleavage by Fe(2+). These findings suggest that cleavage results from binding at a substrate-specific site. Fe(2+) is known to be able to cleave amide bonds in proteins at distances up to approximately 12 A. We conclude that the alpha carbon of glutamine 225 is probably within 12 A of the position of the Fe(2+) ion in the Fe(2+)-tetracycline complex bound to the protein.