Strain Rhodococcus rhodochrous M8 exhibiting high nitrile hydratase (NHase) activity is a promising catalyst for commercial production of acrylamide. With the aim to enhance the biocatalyst activity, regulation of NHase synthesis in this strain has been investigated. The synthesis of NHase and amidase, which also participated in nitriles metabolism in this strain, was induced by isobutyronitrile, acetamide, or urea. At the same time, the addition of glucose, fructose (rapidly metabolizable carbon sources), or ammonium ions to a medium decreased NHase and amidase synthesis. Using Northern blot analysis we demonstrated that these compounds control the expression of NHase at the transcriptional level. The observed effects of glucose and fructose are not related to the decrease in the intracellular concentration of inducer (acetamide), since these compounds cause neither repression nor inhibition of an inducible active system of acetamide uptake. The synthesis of NHase in R. rhodochrous M8 is proposed to be subjected to different regulatory circuits, i.e., induction, carbon, and nitrogen catabolite regulation, operating at the transcription level. We have optimized the growth conditions for R. rhodochrous M8 on the basis of the obtained data. Application of the improved biocatalyst for acrylamide production allows us to obtain the concentrated (38%) solution of acrylamide.