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The effect of carbohydrates, aromatic alcohols, choline and acetylcholine on the biomass production and biosynthesis of choline esterase was studied with Arthrobacter simplex var. cholinesterasus. Fructose was found to be the best carbon source for the biomass accumulation and synthesis of choline esterase. Almost the same amount of the enzyme was produced on media with glucose and maltose as on the medium with fructose though the biomass yield was much lower. On the contrary, the biomass production was higher on media with acetylcholine and ethanol, but synthesis of the enzyme was inhibited. Choline was not assimilated by the culture. Differences in assimilation of glucose and fructose by the culture were found to depend on their concentration and the presence, or absence, of the inductor (acetylcholine) in the medium. Fructose was assimilated by the culture almost completely irrespective of its concentration and the presence of the inductor in the medium. Glucose was assimilated partly, best of all at a concentration of 0.5%. An increase of the concentration to 1% inhibited assimilation of glucose by the organism though had no effect on the biomass production and synthesis of the enzyme. The inductor stimulated assimilation of glucose by a factor of 1.5. Synthesis of choline esterase on the medium with acetylcholine at a concentration of 1% was increased more than twofold upon addition of glucose at a concentration of 0.1%. Biosynthesis of the enzyme rised with glucose concentration though accumulation of the biomass was inhibited. Inhibition of choline esterase synthesis on the medium with acetylcholine as a sole carbon source is due to a lack of energy and the absence of synthesis of carbon compounds which are acceptors of acetyl and methyl groups.