Glucose exerted a concentration-dependent negative regulation on the biosynthesis of cephamycin C by Streptomyces lactamdurans. Formation of the cephamycin precursor delta(alpha-aminoadipyl)-cysteinyl-valine was greatly decreased by excess glucose. The ring-expanding enzyme deacetoxycephalosporin C synthase was strongly repressed by glucose in vivo. Isopenicillin N synthase (cyclase) and isopenicillin N epimerase were not repressed by glucose. However, the activity of isopenicillin N synthase was inhibited in vitro by glucose 6-phosphate, and the activity of deacetoxycephalosporin C synthase was inhibited by inorganic phosphate, glucose 6-phosphate, fructose 2,6-diphosphate and fructose 1,6-diphosphate. The intracellular cAMP content decreased as growth proceeded and remained lower in glucose-supplemented cells than in control cultures. cAMP did not seem to be involved in glucose control of cephamycin biosynthesis.