Mitogens increase prostaglandin synthesis in fibroblasts. In the present studies, transcriptional activation of the prostaglandin G/H synthase isoenzyme 2 (PGHS-2) gene is shown to be responsible for this induction. Transcription of the PGHS-2 gene maximally increased within 15 mm of stimulation, and elevated cellular PGHS-2 mUNA, protein, and prostaglandin synthase activity were detected shortly thereafter. Pulse-chase experiments showed induced isoenzyme 2 is short lived, with a half-life of 22 mm in chicken embryo fibroblasts. Neoplastic transformation of fibroblasts by a variety of oncogenes induced either or both PGHS-1 and PGHS-2 mRNAs in immortalized murine NIH3T3 cells. Some preference of oncogenes such as v-src to induce PGHS2 mRNA has been previously observed. However, in this study exceptions in which v-src did not induce PGHS-2 mRNA were noted. Analysis of independent cell lines transformed by v-fes showed this oncogene induced both PGHS-1 and PGHS-2. Dexamethasone, a synthetic glucocorticoid, selectively decreased both basal and induced levels of PGHS-2 mRNA. Simultaneous addition of the steroid with mitogen reduced mitogen-induced PGHS-2 mRNA levels by 80%. However, nuclear run-on experiments failed to detect any decrease in PGHS-2 mRNA transcription. This suggested dexamethasone rapidly caused PGHS-2 mRNA destabilization. Cycloheximide blocked PGHS-2 mRNA decrease. A fibroblast cell line, RS2, was identified that contained only isoenzyme 2 and possessed PGHS activity that was more highly mitogen-inducible than in any other cell line yet described. Mitogen-inducible activity in RS2 cells was inhibited by aspirin, indicating that PGHS-2, like PGHS-1, can be inactivated by this drug.