Three groups of the nitrogen assimilation cycle enzymes (glutamate synthases (GTS), glutamine synthases (GS), and glutamate dehydrogenases (GD)) were studied in Bacillus subtilis strains with hyperproduction of riboflavin (vitamin B2). It was found that in all strains tested activity of GS was virtually the same, activity of GD was absent, and activity of GTS was reduced. In strains 41 and 24, riboflavin producers, activity of GTS was 30-60% the enzyme activity in the original strain (wild-type RosR). The most pronounced decrease in the activity of GTS (0-12% relative to RosR) was observed in the strain AS5, which had the highest level of biosynthetic activity relative to the other strains. According to the results of determination of the sensitivity of induction of beta-xylosidase to glucose- and fructose-induced catabolic repression, none of the strains studied was characterized by disorders in the protein CcpA, a global regulator of the catabolic repression in gram-positive bacteria, which is required for reducing amination and resulting activation of biosynthesis of glutamic acid in cell. It was suggested that mutations responsible for partial or complete inhibition of GTS biosynthesis caused an increase in the intracellular pool of glutamine. The intracellular pool of glutamine is a nitrogen source for riboflavin in cell. It follows from the results of this work that there is a trend toward an increase in the rate of biosynthesis of vitamin B2 in mutants with inhibited GTS activity. However, the complexity of the processes of regulation of nitrogen assimilation enzymes makes it difficult to find a distinct correlation between GTS activity and riboflavin biosynthesis in these strains.