We have used the technique of DNA-excess filter hybridization to measure directly the content and metabolism of the mRNA for dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). The studies were conducted with a methotrexate-resistant derivative of mouse 3T6 fibroblasts (M50L3) that overproduces the enzyme and its mRNA by a factor of 300 but regulates the level of the enzyme during the cell cycle in the same manner as normal 3T6 cells. We found that, when resting (G0) M50L3 cells were serum-stimulated to reenter the cell cycle, the 10-fold increase in the rate of synthesis of DHFR that occurs at the beginning of S phase was the result of a corresponding increase in DHFR mRNA content. In pulse-labeling experiments, we found that there was a similar increase in the rate of production of the mRNA just prior to S phase. However, the half-life of the mRNA was the same (7.5 hr) in resting and exponentially growing cells. Therefore, the increase in DHFR mRNA content was due to an increase in the rate of production rather than an increase in the stability of the message. The delay between addition of [3H]-uridine to the culture medium and the emergence of DHFR mRNA from the nucleus was 15-20 min for both resting and growing M50L3 cells. A similar delay was observed for total mRNA. Therefore, the time required for the processing of newly synthesized DHFR heterogeneous nuclear RNA into DHFR mRNA is about the same as that for the average mRNA.