Mutants of Escherichia coli defective in the mdoA locus are blocked at an early stage in the biosynthesis of membrane-derived oligosaccharides. The mdoA locus has now been cloned into multicopy plasmids. A 5 kb DNA fragment is necessary to complement mdoA mutations. Cells harbouring the mdoA+ plasmid produced three to four times more MDO than wild-type cells. MDO overproduction did not affect the degree of MDO substitution with sn-1-phosphoglycerol residues. The biosynthesis of MDO remained under osmotic control in overproducing strains.