The method for differential fluorescence staining of cellular RNA and DNA by acridine orange (AO) was optimized for 3T3 and SV40-3T3 cells. Cellular contents of DNA and of ribosomal RNA (rRNA) were determined by dual-channel flow cytometry during cell-density-dependent proliferation and after stimulation of quiescent cells. With increasing density of 3T3 cells, cellular content of rRNA decreases by about 60%, whereas SV40-3T3 cells do not exhibit a significant dependence of rRNA content on cell density. 3T3 cells stimulated early after becoming quiescent resume reaccumulation of rRNA after a delay of only 4 h, whereas cells maintained at quiescence for several days exhibit a delay of about 12 h before a significant rise of rRNA is observed. The extent of rise of cellular rRNA content after different regimens of stimulation of quiescent 3T3 cells does not correlate well with the fraction of cells entering the cell cycle. These and other reported instances of discordance between rRNA content and stimulation into the cell cycle are resolved by showing that of the two signals governing entry into the cell cycle only the progression signal, but not the competence signal is associated with reaccumulation of cellular rRNA. The present results are consistent with the progression function being in essence the achievement of a threshold number of ribosomes per cell, which in conjunction with the competence signal is sufficient for initiation of the cell cycle.