We studied the activities of ribosomal genes (nucleolus organizing regions) at successive stages of cultivation of the mouse R1 embryonic stem cells. The total number and number of active nucleolar organizers were estimated by means of in situ hybridization with mouse rDNA probes and argentophilic staining of nucleolus forming chromosomes regions from the 16th until the 32nd passages. The data we obtained suggest that the total number of nucleolar organizers per metaphase plate was constant (as a rule, eight), while the mean number of active nucleolar organizers progressively increased from the early (16th) to the late (32nd) passages: 5.2 ± 0.4 versus 7.4 ± 0.9 argentophilic organizers per cell. Cell heterogeneity by the number of active nucleolar organizers also increased during the late passages. Taken together, these data suggest activation of ribosomal DNA transcription and synthesis of ribosomes during cultivation of mouse R1 embryonic stem cells. Based on the experimental and published data, it has been proposed that activation of ribosomal genes correlates in time with a decreased capacity of embryonic stem cells for pluripotent differentiation. © 2005 Pleiades Publishing, Inc.