A method of subtractive hybridization using biotinylated DNA and phenol emulsion reassociation technique (PERT) has been proposed. A possibility of combining these techniques has been shown for the first time. The effect of biotinylation degree on the formation of water-insoluble Bio-DNA aggregates was studied. The conditions when Bio-DNA aggregation is actually absent were revealed. A possible use of the above method in hybridization experiments in a wide range of DNA concentrations has been shown. The time of hybridization was 0.5-1 h. The method was approved on a model system, and its possible application for the enrichment of rare mRNA was shown. No less than 300-fold enrichment is achieved for a rare transcript (IL-2) in three cycles of subtractive hybridization.