We have developed a rapid autoradiographic colony assay for detecting mutants with elevated levels of certain biosynthetic enzymes. Four Escherichia coli strains in which the specific activity of the membrane enzyme diglyceride kinase is increased 5-10-fold have been obtained with this approach. The mutant kinase has the same thermal denaturation profile and subcellular localization as the wild type. Five other membrane enzymes involved in phospholipid bilayer assembly are unaffected. In one of these strains (GK-1) the mutation (dgkR-1) responsible for the elevated kinase has been mapped at a new site near minute 92, while the previously identified structural gene (dgk) lies near minute 90. When the structural gene for the kinase (dgk) is cloned on a multi-copy vector-like ColE1, the kinase can be overproduced 5-10-fold on the basis of gene dosage (Lightner, V. A., Larson, T. J., Tailleur, P., Kantor, G. D., Raetz, C. R. H., Bell, R. M., and Modrich, P. (1980) J. Biol. Chem. 255, 9413-9420). Introduction of such hybrid plasmids into a mutant harboring dgkR-1 leads to a multiplicative (rather than additive) effect, resulting in specific activities of diglyceride kinase that are 35-75-fold higher than normal. These results show that dgkR-1 is a trans-acting mutation and suggest the existence of novel regulatory proteins (or metabolites) that direct the expression of certain membrane enzymes.