Background. Bacteria synthesize H₂S by enzymes of defense system orthologous to mammalian cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS) or 3-mercaptopyruvate sulfurtransferase (3MST). Genetic disruption of H₂S biogenesis increases bacterial sensitivity to drugs, and enzymes of defense system may be targets for H₂S biosynthesis inhibition. The aim of the present study was the determining putative G-quadruplexes (G4s) formed by two G-tetrades in promoters and genes of M. bovis defense system encoding enzymes of H₂S biosynthesis. Methods. QGRS Mapper software was used for search of G4s motifs and determination of their G-scores in DNA sense and antisense strands of M. bovis AF2122/97 reference strain [1]. 3D models G4s were predicted with AlphaFold 3 AI (AF3) [2]. Results. A search of G4s in promoters and genes encoding H₂S biosynthesis enzymes of the reference strain M. bovis AF2122/97 with subsequent their estimation and confirmation by 3D modeling was performed. Promoter regions and genes (i) cbs encoding CBS, (ii) cds1 (Rv3684) encoding cysteine desulfhydrase, (iii) metB (Rv1079) encoding cystathionine gamma- synthase, (iv) sseA encoding 3MST were analyzed for the G4s presence. In the cbs gene, four G4s with length of 11—14 nucleotides (nt) were found and characterized by a G-score from 19 to 21. Their 3D models were confirmed by AF3 with a parameter indicating the quality of the prediction: from 70> pLDDT>50 to pLDDT>90. In the cds1 (Rv3684) gene, two G4s with length of 17 nt (G-score = 18, 90>pLDDT>70) and 11 nt (G-score = 21, pLDDT>90) were found. In the promoter of the metB gene (Rv1079), G4 with length of 14 nt was found (G-score = 19, 90>pLDDT> 70 — pLDDT>90). In the promoter of the sseA gene, two G4s (15 nt, G-score = 20, pLDDT>90) were identified. Conclusions. Putative G-quadruplexes of two tetrads were identified in the genes and promoters of genes encoding enzymes of the M. bovis defense system of hydrogen sulfide synthesis. Their structures were confirmed by AF3 with high reliability (pLDDT>90). Determined G4s in the gene promoter regions may be targets for the H₂S synthesis inhibition. Grants. 35.00.03.02 from NAAS of Ukraine.