Gel filtration has been used to study the menaquinone content of Escherichia coli. Menaquinones-6, -7, and -9 have been detected and isolated in addition to the previously encountered menaquinone-8. The principal 2-demethylmenaquinone has been unambiguously characterized as 2-octaprenyl-l,4-naphthoquinone. It is accompanied by the corresponding heptaprenyl derivative. A comparison between the fractionation power of hydroxypropylated Sephadex G-25 and G-50 is made. © 1969, American Chemical Society. All rights reserved.