MIC value of streptomycin resistant M. tuberculosis strains was detemlined using tllernproportional method. Sequence analysis of IpsL and rrs genes was performed using botll tlle radioactivernand non-radioactive cycle sequencing protocols. For functional analysis ofrRNA of M. tuberculosis, M.rnsmegmatis strain mc2 155 was used as a model system. There were 34 streptomycin resistantrnMycobacterium tuberculosis strains subjected for MIC value dete~ation. This included 18 strains fromrnSweden and 16. strains from Etlliopia. Their streptomycin resistance character had been previouslyrnconfirmed by culruring in BACTEC media contailling 4~lg/ml streptomycin. Eleven strains growing onrn7HI0 plates containing ~ 160 )lg/ml streptomycin were classed as high level resistant, 13 strains growingrnon 5-10 )lg/ml streptomycin classed low level resistant and 10 strains were found to be susceptible tornstreptomycin. DNA was extracted from tlle 11 high level and from 10 of tlle 13 low level streptomycinrnresistant strains. The extracted DNA was PCR amplified by targeting the rpsL and rrs genes. PCRrnproducts of all the high level and 6 of the low level resistant strains were sequence analyzed. For thernrpsL gene, 9 of the high level resistant strains had the previously documented mutation at codon 43, andrnone strain has mutation at codon 88. One of the high level streptomycin resistant strains was negative forrnPCR amplification of the rpsL gene. All sequence analyzed low level streptoniycin resistant strains werernwild type for the rpsL and rrs genes. Since the low level resistant strairis did not show any of therndocwnented mutations, it is likely that they nlight have mutations in other ribosomal genes. Tillsrnpossibility was explored by manipulating the mycobacterial relative M. smegmatis for functional analysisrnof the rRNA. M. s111egmatis with oniy one functional rRL'IA operon was generated by replacing thernsecond rRNA operon with plasntid derived inactivated rRNA operon. For replacing one of the rRNArnopeions, a replacement plasntid was constructed. The replacement plasntid contained one rRi'lA operonrninactivated by kanamycin resistant marker. As a counter selectable marker, the plasntid is equipped withrnthe SacB gene which is lethal to mycobacteria in the presence of 15 % sucrose. After transformation,rncolonies were selected on LB plates contailling kanamycin 25)lg/ml and different sucrose concentrations.rnM. smegmatis transformants resulted from double crossover and homologous recombinations werernidentified by their kanamycin and sucrose resistant phenotypes. To check for replacements of one of tllernrRNA operon, transfonnants were analyzed by Southern blotting. Competent cells of M. slllegmatis withrnone functional rRi'lA operon were prepared and transfOlmed with plasntid construct that contained thernwild type rpsL gene. Transfonnants were selected on LB plates contailling 25)lg/ml streptomycin.rnDetermination of MIC value of the streptomycin resistant M. smegmatis transformants and sequencing ofrnrrs, rpsL and the entire genes of the rRNA operon will need to be detennilled. This will be necessary tornestablish tlle mutation induced. If established the hitherto undocumented mutation will be useful to tracernthe mechanisms of resistance in low level streptomycin resistant M. tuberculosis strains.