molybdenum cofactor biosynthesis in escherichia coli requirement of the chlb gene product for the formation of molybdopterin guanine dinucleotide the chlorateresistant mutants of escherichia coli are affected in the biosynthesis of the molybdenum cofactor and show pleiotropic loss of the activities of those enzymes which require the cofactor the molybdenum cofactor in all molybdoenzymes other than nitrogenase is a complex of the metal with a unique pterin termed molybdopterin the molybdenum cofactor in a number of e coli enzymes has been shown to contain gmp in addition to the metalmolybdopterin complex with the gmp appended in pyrophosphate linkage to the terminal phosphate ester on the molybdopterin side chain in this paper we have examined the biochemistry of the chlb mutant and show that the gene product of the chlb locus is essential for the addition of the gmp moiety to form molybdopterin guanine dinucleotide a step which occurs late in the cofactor biosynthetic pathway in e coli sensitive techniques were developed for the identification of fluorescent derivatives of molybdopterin and of molybdopterin guanine dinucleotide in extracts of e coli cells wild type cells were shown to contain both molybdopterin and molybdopterin guanine dinucleotide while cells of chlb mutants were found to contain elevated levels of molybdopterin but no detectable molybdopterin guanine dinucleotide