The effects of productive adenovirus infection on host gene expression were studied by using a line of methotrexate-resistant HeLa cells with amplified dihydrofolate reductase (DHFR) genes. We have previously reported that synthesis of DHFR is induced threefold early in infection and is shut off late in infection (Yoder et al., Mol. Cell. Biol. 3:819-828, 1983). These changes in DHFR protein synthesis are accompanied by changes in both the steady-state cytoplasmic levels of DHFR mRNA and in the rate of appearance of DHFR mRNA in the cytoplasm. In this report, we examined the mechanism of nuclear control of DHFR mRNA levels. Transcription of DHFR-specific sequences continued at a constant rate throughout infection, representing 0.015% of the total transcriptional activity. In contrast, nuclear steady-state levels of DHFR sequences changed in correspondence to the changing rate of appearance of DHFR mRNA in the cytoplasm. That is, nuclear levels of DHFR-specific sequences rose 2.5-fold early in infection and declined to a level below that found in uninfected cells late in infection. Thus, the relative nuclear stability of DHFR sequences changed throughout the course of infection such that during the time of induction, DHFR sequences were preferentially stabilized. This stabilization was transient, however, and was no longer observed by the time of shutoff. These data indicate that posttranscriptional nuclear events are important in the regulation of DHFR gene expression by adenovirus.