All the phage-promoter containing subcloning vectors available for in vitro transcription reactions contain a polylinker away from the transcription initiation site. A new SP6 transcription subcloning vector, pCKSP6, has been constructed, in which a gene can be inserted precisely at the transcription initiation site. This was achieved by bringing the BamHI cleavage site into the initiation site. When DNA ends of both insert gene and BamHI cleaved pCKSP6 are made blunt-ended using a single strand specific nuclease, the in vitro transcripts of the recombinant DNA by SP6 RNA polymerase will contain only the gene sequence immediately after the initiation base G.Mung bean nuclease was used to generate a series of mutants resulting from step-wise deletion of single base pairs around the initiation site. Transcription assays with these SP6 promoter mutants revealed that not only the sequence immediately upstream of the initiation site but also the six base pairs from position +1 to +6 are important elements for promoter binding and/or transcription initiation activity. Furthermore, there appears to be a hierachy of importance of each base pair in the order of position +1 < +2 < +3 < +4, +5, +6, -1, -2.