A new effective method of site-specific mutagenesis in the close vicinity of unique restriction sites of plasmids, based on the use of two oligonucleotide primers, mutagenic and auxiliary, has been suggested. A site-specific insertion of Pribnow box (TATAATG) before promoterless gal operon of the promoter-testing plasmid pHD-001-14-11 has been performed with the yield of mutants up to 95%. Data on the gal operon expression and S1-nuclease mapping of the transcription start point indicate the formation of an active promoter in the region of the insertion.