The molecular assembly of the major outer membrane lipoprotein on the peptidoglycan layer was studied using two hybrid genes coding for different OmpF-lipoprotein hybrid proteins. One gene codes for a "lipoprotein" in which the diacylglyceryl cysteine residue is replaced with the Ala-Glu residue of the NH2 terminus of the OmpF protein (hybrid protein I). The other gene codes for the lipid-free "lipoprotein" from which the COOH-terminal lysine residue was further deleted (hybrid protein II). Hybrid protein I existed as a trimer. A significant portion of it was found to be composed of only the free form, which was noncovalently associated with the peptidoglycan layer. The purified hybrid protein I trimer was dissociated into the subunit in the presence of guanidine-HCl and reassociated on dialysis. Both the native and reassociated trimers were bound to the lipoprotein-free peptidoglycan layer. No enhancement of the binding was observed when the reassociation reaction was carried out simultaneously. Hybrid protein II, on the other hand, did not exhibit association with peptidoglycan in both the cellular fractionation and in vitro binding experiments, although it existed as a trimer. It is concluded that 1) the protein domain of the lipoprotein exists as a trimer which is noncovalently as well as covalently associated with the peptidoglycan layer and 2) although the deletion of the COOH terminal lysine residue did not interfere with the trimerization, it interfered with the noncovalent interaction with the peptidoglycan layer.