355 Escherichia coli mutants requiring riboflavin (RF) for their growth were selected using N-methyl-N-nitro-N-nitrosoguanidine. The mutants were subdivided into four biochemical groups according to their capability to accumulate fluorescent pigments in the medium and their nature, the ability to grow in a medium containing either diacetyl or 6,7-dimethyl-8-ribityl lumazine (DMRL), and the activity of GTP cyclohydrolase II and RF synthase. The rate of RF accumulation by the parent strain of E. coli and the rate of DMRL synthesis by the mutants with blocked RF synthase were identical when the cells were grown in a medium without flavins and did not change when the cells were incubated in a medium containing rifampicin, an inhibitor of transcription. RF had no effect on the rate of DMRL accumulation in the cultural broth when the mutants belonging to the fourth biochemical group were cultivated in a medium with RF. Under the same conditions, RF did not inhibit the activities of GTP cyclohydrolase II and RF synthase in the mutants of the second biochemical group. It is supposed that the enzymes of flavinogenesis in E. coli are constitutive ones.