Synthesis of mature 28-S ribosomal RNA and 60-S ribosomal subunits is inhibited in baby hamster kidney (BHK) cell line ts 422E at non-permissive temperature (39 "C). This leads to a 66 decrease of total ribosomes per cell, a marked imbalance between the large and small ribosomal subunits in the cytoplasm and a decrease of cells per dish after prolonged culture at 39 "C. However, inhibition of ribosome synthesis does not affect progression of cells through the GI period of the cell division cycle, the length of the pre-replicative period, and the rate of entry of cells into S phase. In contrast to culture at non-permissive temperature, culture of BHK ts 422E cells in the presence of 0.04 pg/ml actinomycin D at 33 "C inhibits markedly the entry into S period. It is concluded that low doses of actinomycin D exert their inhibitory effect on cell growth by preventing maturation and transport of mRNA rather than by interfering with ribosome synthesis. Microfluorometric analysis revealed only slight differences in the distribution of BHK ts 422E cells in GI, S and GZ phases of the cycle either when cultured at 33°C or at 39°C. When too few ribosomes per cell are produced in BHK ts 422E cells at 39 "C, cells do not seem to be arrested reversibly at a specific point of the cell cycle but rather to die at random.The nucleolus has been considered to play a prominent role in cell proliferation. There are many reports which demonstrate that the nucleolus is markedly activated in cells stimulated to proliferate. An enhanced synthesis of rRNA in stimulated cells has been shown by an increased incorporation of radioactive precursors into nucleolar [l-51 or cytoplasmic rRNA [6-101 as well as by an increased activity of nucleolar RNA polymerase A [5, 11 -131. On the other hand, selective inhibition of nucleolar RNA synthesis by low doses of actinomycin or by lucanthone at the beginning of the GI period caused a delay of the entry of cells into the S phase [14-171. These findings were interpreted in terms of the dependence of DNA replication upon the contuined supply of ribosomal particles. Since, however, in all these studies only the biosynthesis of pre-rRNA was concerned and neither the processing of rRNA nor the assembly of mature ribosomal subunits has been studied, the question of whether growth stimulation of mammalian cells is necessarily correlated with an increased biosynthesis of ribosomes cannot yet be answered. The purpose of this work was to investigate Ahhreviations. BHK, baby hamster kidney; pre-rRNA, precursor to ribosomal RNA.to what extent, if at all, ribosome formation is a prerequisite for cells to traverse GI and enter S phase. We have used a temperature-sensitive mutant of BHK cells, ts 422E3, that is defective in the processing of 28-S rRNA at the non-permissive temperature [ 18, 191. The results suggest that the initiation of DNA replication is not related to an accumulation of cytoplasmic ribosomes during the GI period. EXPERIMENTAL PROCEDURESCell Culture