Transcriptional regulation following mycobacteriophage I3 infection has been investigated. For this purpose, RNA polymerase mutants (rif)of host bacterium,Mycobacterium smegmatis have been isolated and characterised. Phage growth inrifs and rifr cells in presence of rifampicin revealed the involvement of host RNA polymerase in phage genome transcription. This was confirmed by studies onin vivo RNA synthesis as well as by direct RNA polymerase assay after phage infection. Significant stimulation in RNA polymerase activity was seen following phage infection. The maximal levels were attained in about 60 min post infection and maintained throughout the phage development period. The stimulation of polymerase activity was most pronounced when the phage DNA was used as the template. RNA polymerases from uninfected and phage-infectedMycobacterium smegmatis have been purified to homogeniety. The enzyme purification was accomplished by a rapid procedure utilising affinity chromatography on rifampicin-Sepharose columns. Subunit structure analysis of the purified RNA polymerase from uninfected and phage-infected cells showed the presence of α,β, β′ and σ subunits similar to the other prokaryotic RNA polymerases. In addition, a polypeptide of 79, 000 daltons was associated with the enzyme after phage infection. The enzymes differed in their properties with respect to template specificity. Phage 13 DNA was the preferred template for the modified RNA polymerase isolated from infected cells which may account for the transcriptional switch required for phage development.