Ribonucleotide reductase in mammalian cells is composed of two nonidentical subunits, proteins R1 and R2, each inactive alone. The R1 protein is present in excess in proliferating cells, and its levels are constant during the cell cycle. Expression of the R2 protein, which is limiting for enzyme activity, is strictly S-phase-correlated. In this paper, we have used antisense RNA probes in a solution hybridization assay to measure the levels of R1 and R2 mRNA during the cell cycle in centrifugally elutriated cells and in cells synchronized by isoleucine or serum starvation. The levels of both transcripts were very low or undetectable in G0/G1-phase cells, showed a pronounced increase as cells progressed into S phase, and then declined when cells progressed into G2 + M phase. The R1 and R2 transcripts increased in parallel, starting slightly before the rise in S-phase cells, and reached the same levels. The relative lack of cell cycle dependent variation in R1 protein levels, obtained previously, may therefore simply be a consequence of the long half-life of the R1 protein. Hydroxyurea-resistant, R2-overproducing mouse TA3 cells showed the same regulation of the R1 and R2 transcripts as the parental cells, but with R2 mRNA at a 40-fold higher level.