Transcription of the junB gene is rapidly and transiently induced following stimulation of susceptible cells by growth factors in serum. Our previous studies demonstrated that serum inducibility of junB and other immediate-early genes is markedly attenuated in fibroblasts chronically transformed by the viral Src oncoprotein. Moreover, attenuation of junB induction occurs at the transcriptional level. To characterize further the molecular mechanisms of this attenuation, various full-length and recombinant junB constructs were transfected into normal and viral Src-transformed rat fibroblasts. A stable transfection system was used to faithfully reproduce regulation of the junB gene. Analyses of pooled populations of stably transfected cells demonstrate that sequences between -89 and +32 (relative to the transcriptional start site) are sufficient to confer both serum inducibility of the junB gene in normal cells and its attenuation in viral Src-transformed cells. By contrast, attenuation of c-fos serum inducibility by Src transformation involves element(s) distinct from regulatory elements identified previously in the c-fos promoter. These results identify a proximal promoter region of the junB gene that is involved in a novel negative regulation of its transcription.