Most bacterial outer membrane proteins contain a phenylalanine at their C terminus. It has been shown that this residue has an important role in the efficient and correct assembly of PhoE protein into the Escherichia coli outer membrane, since its substitution or deletion resulted in the accumulation of trypsin-sensitive monomers of this normally trimeric protein. Here, the role of the C-terminal Phe in the assembly of PhoE was studied in further detail. Immunocytochemical labelling on ultrathin cryosections revealed that a mutant PhoE protein that lacks the C-terminal Phe accumulates in the periplasm. However, when the expression levels of the altered species were reduced, the efficiency of outer membrane incorporation was increased and the lethal effects were alleviated. The role of the C-terminal Phe in protein folding, trimerization and outer membrane incorporation was further studied in vitro. Deletion of this residue interfered with the efficiency of the formation of an assembly-competent folded monomer, and the stability of this PhoE form was affected. The in vitro trimerization and insertion into outer membranes were not affected by the mutation.