The nucleolar protein SURF-6 is an evolutionarily conserved and vitally important eukaryotic protein; however, its function in mammals still remains unclear. In the present study, we have examined the effect of SURF-6 overexpression on the content of different pre-rRNA sequences in mouse NIH/3T3 fibroblasts. The overexpression of SURF-6 was induced by a Tet-On system of regulation of gene expression and two plasmid constructs. Plasmid pUHrT62-1 encoded the rtTA protein, a tetracycline-dependent factor of transcription activation, and plasmid pBI-SURF6 contained the coding sequences of the genes of the mouse SURF-6 protein and EGFP, whose expression was controlled by the rtTA-dependent promoter. An increase in the SURF-6 content was observed 24 and 48 h after the addition of the inductor doxycycline. The dot hybridization of total RNA from cells having an increased SURF-6 content with biotinylated oligonucleotide probes to different regions of mouse primary pre-rRNA transcripts revealed a sevenfold increase in the content of ITS2 and a twofold increase in the content of 5′ETS compared with their levels in cells transfected with the same plasmids but incubated without doxycycline. The content of fragments corresponding to 18S, 5.8S, and 28S rRNAs remained almost unchanged. These observations showed for the first time that the mammalian nucleolar protein SURF-6, similar to its homolog the yeast protein Rrp14, contributes to the stabilization (prevents the degradation) of pre-rRNA intragenic transcribed spacers. At present, no metazoan proteins that play a similar role in ribosome biogenesis have been described. © 2010 Pleiades Publishing, Ltd.