Levels of mRNA for IFN-beta 2/B cell differentiation factor2/hepatocyte-stimulating factor (IFN-beta 2) in confluent quiescent cultures of human diploid fibroblasts (FS-4 strain) are enhanced by TNF, IL-1 alpha and beta, platelet-derived growth factor (PDGF) and IFN-beta 1. Of these cytokines, IL-1 alpha and beta cause a particularly strong increase in the accumulation of IFN-beta 2 mRNA in fibroblasts. We have evaluated whether the IFN-beta 2 gene is regulated at the transcriptional level by using nuclear run-on transcription assays. We observed that the IFN-beta 2 gene is transcribed at a low level in uninduced FS-4 cells and that this transcriptional activity is increased 2- to 3-fold in cycloheximide-treated cells, 20- to 35-fold in IL-1 alpha-treated cells, and 5- to 15-fold in TNF-treated cells. PDGF and IFN-beta 1 enhance transcription across the IFN-beta 2 gene 2- to 3-fold. The enhancing effect of IL-1 alpha on IFN-beta 2 gene transcription, but not that of TNF, PDGF, or IFN-beta 1, is inhibited by cycloheximide, suggesting that newly-synthesized protein is involved in the increase in IFN-beta 2 transcription in response to IL-1 alpha but not in the response to the other stimuli. Furthermore, the enhancement of IFN-beta 2 transcription is sustained for up to 14 h after IL-1 alpha induction but is transient and declines to base line levels within 6 h after TNF addition. These observations suggest that there are important differences in the mechanisms by which IL-1 alpha and TNF increase IFN-beta 2 gene transcription in fibroblasts.